Selective Isolation and Characterisation of Members of the Family Streptosporangiaceae

Agrawal, P. and M.Goodfellow Department of Microbiology, Medical School, Newcastle upon Tyne, UK

Dry heat treatment of air-dried soil samples, additional pretreatment regimes and dilution plate culture with selective synthetic media (Nonomura and Ohara, 1988. In: Biology of Actinomycetes '88 [Y.Okami, T.Beppu and H.Ogawara, eds.]. Jap. Sci.Soc. Press) were used to selectively isolate Microbispora, Microtetraspora and Streptosporangium strains. Members of all three genera were isolated on AV and HV agars where 10^-1 soil dilutions had been treated with either yeast extract (6%, w/v) or sodium dodecylsulphate (0.06%, w/v) for 20 minutes at 40 C. Corresponding dilutions treated with phenol (1.5%, w/v) at 30 C for 30 minutes prior to plating out proved selective for Microbispora and Microtetraspora strains. The average counts (per gram dry weight soil) of the target organisms on HV agar were: a) Microbispora -1.8xl0^4 (yeast extract), 3.1x10^4 (SDS) and 5.2x10^4 (phenol); b) Microtetraspora - 2.8x10^4 (yeast extract), 3.0x10^4 (SDS) and 7.0x10^3 (phenol), and c) Streptosporangium - 8.5x10^4 (yeast extract), 2.1x10^4 (SDS) and 0 (phenol). Selected isolates were characterised, notably by using fluorogenic enzyme probes based on 7-amino-4-methylcoumarin and 4- methylumbelliferone.

Specific Serodiagnosis of Nocardia Infection

Boiron P.

Institut Pasteur, Unite de Mycologie, Paris Cedex, France

Nocardiosis is an opportunistic infection in patients with an impaired host defense mechanism, though infection in normal hosts has also been reported. Nocardiosis is difficult to recognize on the basis of clinical, radiological, or histological findings. A definitive diagnosis depends on the isolation and identification of a Nocardia sp., a procedure which can take 2 to 3 weeks. However, early recognition of nocardiosis, even by aggressive, invasive diagnostic efforts, is often necessary, since specific antibiotic therapy improves the prognosis. The need for a reliable serological test to detect specific antibodies in the sera of nocardiosis patients and to minimize the need for invasive diagnostic procedures is therefore obvious.

Past experience with serological methods to diagnose nocardiosis in animals and in man have met with somewhat limited success. The data support the suggestion that the infected individuals can be identified but that the high degree of crossreactivity seriously hinders the specificity of these tests.

We take advantage of the high degree of crossreactivity between nocardiae and mycobacteria to purify nocardiae- specific antigens by immunoaffinity chromatography and to investigate the usefulness of western blot (immunoblot assay) in the specific diagnosis of Nocardia infection.

A specific immunodominant 54kilodalton (kDa) antigen purified from the culture filtrate of N. asteroides by immunoaffinity chromatography accompanied by two others of 31- and 62kDa. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. The western blot technique was applied to detecting the immunologic response to nocardiae. All serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to the 54-kDa antigen with all serum sample obtained from patients with tuberculosis, except one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls.

The role of the humoral immune response to infection with Nocardia spp. is not well understood. The availability of a suitable antigen may further our understanding of the pathogenesis of nocardiosis. The 54-kDa protein is a candidate for use as a probe to study the humoral immunologic response to nocardiae.

The Use of Ribosomal Rna Sequences for the Identification of Members of the Family Pseudonocardiaceae

Bowen,T., S.Warwick, J.Challans and M.Embley

Microbial Technology Research Unit, Polytechnic of East London, London, UK

Ribosomal RNA (rRNA) has been sequenced from all of the currently recognised species and genera in the family Pseudonocardiaceae. Representatives of newly described genera such as Actinokineospora and Saccharothrix have also been included. The results of these analyses give a clear picture of the phylogeny of the Pseudonocardiaceae. Oligonucleotide probes have been devised to variable regions of 16S rRNA and used to identify members of the family. A small scale method for DNA extraction has been used on single colonies to provide material for the amplification of rRNA genes by the polymerase chain reaction. The amplified products were used as targets for evaluating probes. The amplification products were also used as the starting material for asymmetric amplification for direct sequencing of RNA genes. The implications of this work for the identification of members of the family Pseudonocardiaceae will be discussed.

Isolation and Characterisation of Streptomycetes Implicated in Common Scab of Potatoes

Bramwell, P.A. and E.M.H.Wellington

Department of Biological Sciences, University of Warwick, Coventry, UK

Interests in our laboratory are concerned with following the fate of microorganisms, in particular the streptomycetes, in the natural environment. An essential aspect to this work being the development of reliable methodology for isolation and characterisation of microorganisms from environmental samples.

Common scab, an agronomic disease present in potato growing regions throughout the world, results in the formation of superficial lesions on the tuber surface reducing the market value of the crop.

The group of organisms associated with common scab was identified as early as 1891 by Thaxter, and recognized as Streptomyces scabies Waksman et Henrici, in 1943. Opinions on the nature of this group continue to differ, from those who support the view that the streptomycetes able to cause scab form a closely defined group i.e. Streptomyces scabies, to those who find that a wide range of streptomycetes are implicated in the disease.

The characterisation of known pathogenic isolates from various agricultural centres indicated that streptomycetes from a range of cluster groups cause scab, as defined using the probabilistic identification matrix of Williams et al., 1983.

Current experimental work involves the use of plant soil microcosms to ascertain the effects of potato plants on streptomycete population levels and the incidence of genetic exchange via conjugative plasmid transfer.

Traditional methods of characterisation are of limited value in the type of seed and recovery experiments required for studying inoculants in situ, against a background microflora of phenotypically similar streptomycetes. Strategies for monitoring the interaction of streptomycete inoculants with the potato surface have included the development of marked strains harbouring the catechol 2,3 dioxygenase, xylE reporter gene and the development of strain- and species specific 16S ribosomal RNA probes.

Development of Regimes for Efficient Recovery of Saccharomonospora viridis Released Into A Contained Compost System

Brooks, R.C., T.R. Fermor* and A.J. McCarthy

Department of Genetics and Microbiology, The University, Liverpool and *Institute of Horticultural Research, Littlehampton, UK

Saccharomonospora viridis strain 125 is a stable lilac-pigmented variant of this species, which is being used as a traceable marker organism in the development of a model compost system for studying release of genetically engineered microorganisms (GEMs). Considerable improvements in the efficiency of isolation of Saccharomonospora viridis from a range of environments have been made, largely through modification of the isolation medium (Amner et al., Appl. Environ. Microbiol., 54, 3107, 1988; Ibidem, 55, 2669, 1989). The purpose of this work was to consider other modifications to isolation procedures with particular emphasis on their practical application to monitoring populations in large-scale composts. A containment facility holding 40 Kg of mushroom compost under regulated environmental conditions has been designed. Containment efficiency is monitored by Andersen air sampling for Sam.viridis BD125 spores.

Experiments on an intermediate scale (800g compost) have resulted in the immediate recovery of 70-80% of spore inoculum from sterile compost, and colonisation of this substrate for up to 7 days. In untreated mushroom compost, recovery efficiencies decrease to 50-60%, largely due to inhibition of growth on isolation plates by Bacillus spp. Some improvement in recovery has been achieved by addition of Chloroquine (900 ml^-1 final concentration) and Teepol (0.01% final concentration) to the diluent. Thus, Sam.viridis BD125 detection limits have been improved from 10^5 to 10^3 cfu g-l compost. Furthermore, experiments over 30 days have shown that strain BD125 becomes incorporated into the indigenous Sam. viridis population when released at high inoculum levels.

Statistical analysis on platings, dilutions and samples all in triplicate, together with considerations of practicability led to design of a sampling regime in which variance was minimised. Results of studies on the relationship between inoculum size and establishment of a stable Sam.viridis BD125 population in compost will also be reported. The degree of congruence between data on composts prepared in laboratory- and large-scale will now be assessed. Finally, progress on the construction of genetically marked strains for studying plasmid survival and transfer in composts will be reported.

R.C. Brooks acknowledges NERC for the award of a CASE studentship and for financial support to attend this meeting.

Problems in the Isolation and Identification of Streptoverticillia

Cardinali, S., G.Firrao and R.Locci

Chair of Mycology, The University, Udine, Italy

In order to evaluate the ecological status of microorganisms belonging to the genus Streptoverticillium, several soil samples from Friuli were screened according to selective isolation methods proposed in the literature. Because the recovery of Streptoverticillium isolates was unsatisfactory, the isolation methods were investigated. Culture collection strains belonging to the genera Streptomyces and Streptoverticillium were examined to test oxytetracycline and lysozyme effectiveness and to identify novel selective inhibitors, chosen on the basis of numerical studies.

Preliminary investigations indicated the selective action of lysozyme and neomicin, whereas oxytetracycline at the suggested concentration appeared to inhibit both Streptomyces and Streptoverticillium. Carbon and nitrogen sources seemed to have no effect.

Using this information 60 soil samples were screened; in the course of the operation 46 Streptoverticillium strains and several hundred Streptomyces strains were isolated. All Streptomyces strains were lysozyme- resistant and 32 of them, chosen as representatives, were assigned to cluster-group A (Williams et al., J. gen. Microbiol., 129, 1743, 1983). The isolation of Streptoverticillium strains seemed to be erratic and not reproducible. All the strains collected were identified by probabilistic methods, and most of them were assigned to clusters 2 and 3 of Locci et al. (Ann. Microbiol., 31,115, 1981). One strain was assigned to cluster 1 and one to 7 while 18 strains remained unidentified. Twelve strains obtained from other workers were recovered in cluster 3.

The strain pattern, submitted to the chi square test, significantly differed from that assessed in a comprehensive taxonomic study of the genus (Locci et al., l.c.). The lack of effective selective isolation methods still impairs ecological studies of streptoverticillia in different habitats.

The Effects of Genetic Manipulation on the Phenetic Identification of Streptomyces Species.

Clarke, S.D., S.T.Williams and DA.Ritchie

Department of Genetics and Microbiology, The

University, Liverpool, UK.

The genus Streptomyces exhibits wide natural diversity and has been difficult to define taxonomically. A comprehensive numerical classification of Streptomyces by Williams and his coworkers (Williams et al., J. gen. Microbiol., 129, 1743, 1983) provided a more reliable classification system, upon which a probabilistic identification matrix for Streptomyces was created (Langham et al., J. gen. Microbiol., 135, 121, 1989). Recent advances in Streptomyces genetics has lead to the production of a large number of genetically manipulated strains. Many such manipulated strains are of great industrial importance, and the question of their taxonomic identity has arisen, with particular regard to strain patents.

Aiming to help resolve this dilemma, genetically manipulated strains from a number of sources have been identified along side their original parental strain, using the probabilistic identification matrix. All but one of the genetic manipulations studied were capable of altering a strain identification. None of the altered identifications were however, positively identified to an alternative species cluster group to that of the parent strain. A study of the stability of the characters in the identification matrix and that of species cluster groups were made with respect to various genetic manipulations.

Considering the problems of classifying Streptomyces a preliminary investigation was undertaken into the use of restriction fragment length polymorphisms (RFLP's), as a rapid and accurate taxonomic tool in Streptomyces. DNA's from selected Streptomyces species were restricted and probed with a 16S and 23S rRNA probe and the digest profiles were assessed taxonomically using the simple matching coefficient. Strain similarities were compared with those obtained with DNA-DNA hybridisation studies and numerical classification.

A Student-friendly Method for Isolating Actinomycetes From Soil Which Employs Agrochemical Fungicides

Cross, T., G.Alderson and A.Hicks

Department of Biomedical Sciences, The University, Bradford, UK.

Our Objectives were (1) to devise a simple isolation method for use by students to isolate actinomycetes from soil so that they can safely explore the morphology, enzymatic activities, and biosynthetic capabilities of non pathogenic microbes, and (2) to screen commercially available fungicides to discover one that might replace the essential but expensive antibiotics specified in the majority of recommended isolation media.

Method: (a) dry the soil sample for at least 7d at room temp. (b) grind soil with pestle and mortar (c) sieve soil through mesh size >1 mm (d) weigh 1 g of soil into 9ml sterile tap water (e) shake suspension vigorously (f) stand 5 min (g) transfer 2 drops of supernate with a sterile Pasteur pipette to 100ml sterile tap water agar (1.0% w/v) cooled to 55 C and roll the bottle to mix contents without introducing bubbles (h) hold at 55 C for 5 min (i) pour the molten agar to form a thin layer (>2-3mm) on the surface of pre-poured plates of isolation agar (j) when set incubate plates at 25 C for 14d. Suitable isolation media include Czapek agar (Oxoid), actinomycete isolation agar (Difco), starch casein nitrate agar (Kuster and Williams, Nature, 202, 928, 1964) glycerol arginine agar (El-Nakeeb and Lechevalier, Appl. Microbiol., 11, 75, 1963).

Fungicides: Eight fungicides were compared with cycloheximide and nystatin as possible isolation medium constituents to suppress the growth of fungi. Storite^T (thiabendazole) at 50ppm gave equivalent control of fungi and a cheaper isolation medium. The results suggest that alternative agricultural (e.g. penconazole) and medical azole fungicides (e.g. ketoconazole and the newer triazoles) might also be useful.

Spore-dome Actinomycetes Are Strains of the Genus Kineosporia for Which We Now Suggest A Novel Method of Spore Formation

Cross, T., Gjlderson and M.Eke

Department of Biomedical Sciences, The University, Bradford, UK.

The vegetative hyphae of spore-dome actinomycetes have a wall peptidoglycan containing LL-diamino-pimelic acid (DAP), as described for Kineosporia, (Pagani and Parenti, Int. J. Syst. Bact., 28, 401, 1978), but spore walls contain meso-DAP. Additional chemotaxonomic characters for the genus include a mol % G+C for DNA of 68.4 and a PII phospholipid pattern.

The first spore to be delimited by a double wall at the hyphal tip within a sheath (sporangium) has a wall continuous with the hypha. Maturation of that spore is followed by a succession of new (meso-DAP) spores which form at the cross wall and are surrounded by new sheath layers. Spores formed in water quickly become motile but those accumulating in the slimy matrix at the surface of a colony can remain in file as originally described by Willoughby (Nova Hedwigia, 18, 45, 1969) and later noted by Itoh et al. (Int. J. Syst. Bact., 39, 168, 1989).

Competition Between Streptomycetes in Soil

Eccleston, J.D., S.T.Williams* and A.M.Mortimer

Department of Environmental and Evolutionary Biology and * Department of Genetics and Microbiology, Liverpool, U.K.

The extent of competition within one and between two Streptomyces species (S. cyaneus (A) & S. halstedii (C)) have been studied.

In vitro experiments utilized Petri dishes containing 15 ml medium (pH 7) while 1 gram sterile soil samples (pH 6.8) were employed for the in vivo investigations. Each system was inoculated with a range of initial spore concentrations (co) and incubated (14 days @ 25 C in vitro; 28 days @ 10 C in vivo).

The total number of spores produced remained constant for each system, irrespective of co, indicating the maximum capacity available for spore production in each case. The number of spores produced per colony was found to be density dependent. In vitro, the curve was level at co = 0 to 10 c.f.u./plate, followed by a linear decrease. The extent of this plateau was increased in vivo (co = 0 to 1.8x10^2 (A); 0 to 1.8x10^4 (C) c.f.u./g soil) but the viability of the spores was greatly reduced. During these plateux little interference occurs between individual colonies, and in vivo results imply that the spores may be better spaced in separated microsites. At higher co values, interference is more likely to occur both physically (touching colonies) or via competition for limited nutrients. A appeared to compete more within itself (steeper gradient) when compared with C.

Competition between the two streptomycetes at various concentrations was examined. In vitro, A perceived C as being only 0.0066 x its equivalent and was unhampered by its presence until the concentration of C reached 1.8x10^4 c.f.u./plate. C perceived A as being 9.35 x its equivalent and a depression in the curve was evident when the concentration of A was only 6 c.f.u./plate. A therefore appeared to be a highly competitive species under these conditions. In vivo, limited competition seemed to occur since both curves were not depressed until the concentration of the competing species was approx. x 10^5 c.f.u./g soil. This seems to indicate that the competitive natures of the streptomycetes were buffered by the heterogeneity of the soil, the lower nutrient status and the lower temperature.

A New Method for the Extraction of Streptomycete

Spores From Soil.

Herron, P.R. and E.M.H.Wellington

Department of Biological Sciences, University

of Warwick, Coventry, U.K.

The recent interest concerning the release of genetically engineered microorganisms into the environment has highlighted the need for new approaches to their recovery and enumeration. Traditional methods of soil bacterial extraction can give detection limits of ca. 10^2 cfu/g, it is felt that, in any release into the field, such an organism would eventually drop to a titre much lower than this. Thus, we have developed a method that can detect 10 streptomycetes/100g sterile soil and can enumerate 1000 in 100g sterile soil. This method makes use of an ion-exchange resin to disperse soil particles, followed by successive rounds of centrifugation to concentrate bacterial cells. In addition to being very sensitive to the numbers of streptomycetes in soil this procedure also preferentially concentrates spores rather than mycelia and thus can give an incite into the state of the organism in soil.

The Abundance and Diversity of Micromonospora in Soil and Sediments.

Hodges, J.A. and E.M.H.Wellington

Department of Biological Sciences, University of Warwick, Coventry, U.K.

The majority of Micromonospora are aerobic and filamentous, monosporic actinomycetes. As the second most abundant group within the actinomycetes they occupy an important niche in sediments, soil and aquatic environments. The analysis of physiological and biochemical data such as antibiotic resistance and enzyme profiles has provided valuable information for taxonomic grouping of Micromonospora species and assisted in the production of a probabilistic identification matrix for rapid characterisation. This has provided a direct means of determining the distribution and diversity of Micromonospora in soil and sediments. In addition successful methods for selectively isolating and enumerating Micromonospora have been developed. This information has provided us with the basis for developing additional techniques to further study the diversity within the genera.

Development of Nonantibiotic Resistance Marker Genes for Streptomyces

Kochte-Clemens, B., Ujtauland and J.Cullum

LB Genetik, Universitiit, Kaiserlautern, FRG

It is important to develop marker genes to monitor release of recombinant organisms and antibiotic resistance genes are undesirable because of possible ecological and health problems. The agarase gene of Streptomyces coelicolor A3(2) is a promising candidate because agarase genes are very rare in Streptomyces species and this gene did not hybridise to a range of species tested (Miyashita, Wellington and Cullum, unpublished results). Two possible applications of the agarase gene are possible: (1) Introduction into foreign species to mark recombinant DNA. This could either be on plasmids to investigate stability and transferability or integrated into the chromosome to provide a potentially stable marker. (2) The use of Streptomyces coelicolor A3(2) derivatives that have ngt been subjected to in vitro genetic manipulation as a model system to devise methodology for monitoring release of organisms.

We are investigating ways of obtaining stable integration of the agarase gene in foreign chromosomes. A simple strategy is to introduce it on a temperature-sensitive-replication plasmid and to select for natural integration mecha' nisms by raising the temperature - initial experiments with S. lividans 66 have been promising.

An attractive method of detecting sequences in the soil is using the Polymerase Chain Reaction. We have synthesised oligonucleotide primers for the agarase gene and are investigating their application.

Use of Polyvalent Streptophage for the Selective Isolation of Non-streptomycete Actinomycetes From Soil

Kurtboke, D.I. and S.T.Williams

Department of Genetics and Microbiology, The University, Liverpool, U.K.

The continuing search for new antibiotics and other useful secondary metabolites is of great importance in terms of potential pharmaceutical or industrial applications. Actinomycetes have long been regarded as the principle source of antibiotics with the genus Streptomyces excelling in its capacity for antibiotic production. Since most of the antibiotics and useful secondary metabolites from the common genera have for the most part already been studied, identified and applied in industry, the search for novel compounds has been concentrated on other non-streptomycete genera. However, most rare genera require more specific isolation procedures to detect them (Williams and Vickers, 1988).

Actinophage have been used to investigate relationships between different genera of the Actinomycetales (Bradley et al., 1961; Prauser and Falta, 1968). Since the results of phage research correspond well to the results of biochemical and molecular biological studies (Lechevalier et al., 1986) phage susceptibility has been currently accepted and shown to be an effective and inexpensive aid to taxonomy of many novel genera since during adsorption to receptors, that are usually present in only a very limited number of closely related bacterial strains, phage usually respect host genus boundaries (Prauser, 1984). However, this susceptibility has never been used for selective isolation purposes. Using the highly polyvalent characteristics of streptophage for the members of the family Streptomycetaceae (Prauser, 1970) a new method for the selective isolation of nonstreptomycete actinomycetes was developed.

In this new method, phage susceptibility of streptomycetes have provided a selective means of reducing streptomycetes on isolation plates. With the application of high phage titers, the detection of other non-streptomycete genera was facilitated, since the space left after streptomycete reduction was compensated by non-streptomycetes. When the true lysis occurred streptomycete colonies did not have chance for development during the long incubation which is required for slow growing nonstreptomycete genera. High reduction ratios for streptomycetes were obtained after eradication of bacteria which inhibit free interaction of phage to streptomycetes. After the reduction of streptomycetes and bacteria on the isolation plates, the numbers of fast-growing non- streptomycete genera increased. However, certain selective pressures are also needed to activate dormant spores of slow- growing rare genera whose isolation frequency by conventional methods is lower.

Use of Actinophage for Selective Isolation Purposes Current Problems

Kurtboke, D.I. and S.T.Williams

Department of Genetics and Microbiology, The University, Liverpool, U.K.

Until recently, inaccurate classification of many actinomycete taxa has hindered not only the development of efficient selective isolation procedures (Goodfellow and Williams, 1986) but also the establishment of phage typing systems for reliable identification of actinomycete species or genera (Prauser, 1984). Consequently the use of phage as a natural tool has not been accepted as an effective and inexpensive aid to taxonomy of many novel genera and has not been exploited for selective isolation purposes. Recently it has been shown that streptophage, through their advantageous specificity to the genera or chemotaxonomic groups of the family Streptomycetaceae, can be used for selective isolation of nonstreptomycete actinomycetes from soil (Kurtboke, 1990). The genus Streptomyces has been extensively studied and its definition has been facilitated by phage activity spectra (Wellington and Williams, 1981).The substantial amounts of data about streptophage was the base for the development of a new method for streptomycete reduction. However, before this principle can be extended to other isolation targets, more information is required about the taxonomic level of actinophage activity. This will depend on the number of members of each taxon studied and the number and selection of phage applied, since almost no phage is virulent for all strains of taxon under study (Prauser, 1970). For most other actinomycete genera actinophage specificity requires further study and isolation of more phage banks. For some established genera such as sporoactinomycetes of cell wall chemotype III, no phage has been located despite repeated attempts at isolation (Prauser, 1984). For the members of some genera, such as for Arthrobacter globiformis, phage were also rarely detected unless the soil was nutritionally amended and incubated (Casida and Liu, 1974). Monospecificity of some genera hindered the isolation of their phage and consequently the availability of the adequate knowledge about the activity of the latter at taxonomic level. Also the relatively small number of strains of Nocardioides, Oerskovia and Promicromonospora isolated in different laboratories may indicate their rareness, and compound the difficulties of detecting their phage in soil. Prauser (1984) suggested that either isolation techniques have been inappropriate for these actinomycetes or hosts of a taxonomic position different, from those described in literature that occur in soil.

Adequate knowledge about the taxonomic level of actinophage activity and about phage-host interactions in their natural substrates may aid and encourage the extension of the principle of using actinophage for selective isolation purposes.

Identification of Strains Intermediate Between Rhodococcus and Corynebacterium

Meyer, J.

Zentralinstitut fur Mikrobiologie und Experimentelle Therapie, Jena, DRG

A group of strains isolated from natural substrates (sewage, dairy waste water, liquid manure, leaf litter), containing mycolic acids of short chain length and lacking susceptibility to Rhodococcus phages, was studied biochemically (% G+C of DNA, menaquinones, fatty acid pattern and cell wall acyl type).

The results partially indicate a similarity with Rh.equi, Rh.maris or Rh.luteus, however question the delimitation between Corynebacterium and Rhodococcus.

The Use of 16s Rrna Oligonucleotide Probes for the Detection of Streptomycetes in Soil.

Mullins,P^1, D.Hahn^1' E.M.H.Wellington^1, A-Akkermans^2 and H. Gurtler^3

^1 Department of Biological Sciences, University of Warwick, Coventry, UK,
^2 Department of Microbiology, Wageningen, The Netherlands
and
^3 Novo Industries, Bagsvaerd, Denmark
The analysis of 16S rRNA catalogue and sequence data has provided valuable data for phylogenetic groupings of many actinomycete genera and assisted in their classification. Detailed analysis of sequence data derived either from DNA or RNA has shown the existence of both genus- and species- specific sequences. The use of oligonucleotide probes to detect these sequences has already proved extremely useful in the rapid identification of medically important genera such as Mycobacterium and Actinomyces. They have also been used for ecological studies on groups that are difficult to culture such as Frankia. We have been investigating the usefulness of such probes for studying the ecology of streptomycetes in soil. Initial studies have used genus- and species specific probes for the Streptomyces as sequence data is available for this group. The aim of the current work was to provide a rapid screening of soil isolates to detect strains which resembled streptomycetes and hybridized with a genus specific probe. Other probes have also been used which are more specific and provide a means of detecting certain streptomycete cluster groups. It is also possible to isolate RNA from soil and probe this using dot blots to detect the presence of certain genera and species in soil. This provides a direct means of determining the distribution of streptomycetes in differing habitats. The specificity of these probes has been thoroughly investigated. The ecological applications of 16S rRNA probes for detection and identification of Streptomyces and selected species both in soil and on isolation plates has been investigated.

Selective Isolation of Some Major Species Groups of Nocardia. Rhodococcus and Amycolate, Wall Chemotype Vv (a-wciv) Actinomycete Groups: A Taxonomic Approach.

Parekh, N.R., S.T.Williams and J.C.Vickers*

Department of Genetics and Microbiology, The University, Liverpool and *Glaxo Group Research Ltd., Greenford, U.K.

The multiplicity of approaches to the selective isolation of actinomycetes (Cross, Dev. Ind. Microbiol., 23, 1, 1982; Nolan and Cross, In: Actinomycetes in Biotechnology. Academic Press. London, 1, 1988; Wellington and Cross, In: Progress in Industrial Microbiology. Elsevier, Amsterdam, 17, 7, 1983; Williams and Wellington, In: Bioactive Microbial Products: Search and Discovery. Academic Press. London, 9, 1982) show clearly that differences in the nutritional, physiological and antibiotic sensitivity profiles of different groups can be exploited for the isolation of particular taxa from natural habitats. Recent numerical taxonomic studies have produced extensive data on nutritional and growth affecting characters of Nocardia (Hookey, Ph.D Thesis. University of Newcastle, 1983), Rhodococcus (Goodfellow and Alderson, J. gen. Microbiol., 100, 99, 1977-122) and amycolate wall chemotype IV actinomycetes (Mattinson-Rose, Ph.D Thesis. University of Newcastle, 1986) belonging to 15,10 and 11 major (77.5% Ssm similarity level) species groups respectively. Information abstracted from similar data matrices has already been successfully used in the isolation of other actinomycete taxa, the most comprehensive studies having been carried out on streptomycetes (Vickers et al., In: Biological, Biochemical and Biomedical Aspects of Actinomycetes. Academic Press, London, 553, 1984; Williams et al., In: The Microbe. Cambridge University Press, part II, 219, 1984). Information from the appropriate numerical taxonomic studies was used as a logical basis for the formulation of new media, selective for the isolation of particular species groups of Nocardia, Rhodococcus and a-wdV actinomycetes. The principles and preliminary results of this approach are outlined here.

New selective isolation media were formulated with the assistance of the DIACHAR program (Sneath, Computers and Geosciences, 6, 21, 1980) which selects the most diagnostic characters for individual clusters within a numerical data matrix as well as indicating the degree of differentiation of each particular species group. The program was thus used to select nutritional or tolerance characters which could usefully be incorporated into new selective isolation media. These objectively devised sets of media, when applied together will provide a more accurate picture of the qualitative nature of soil actinomycete populations rather than the 'general' isolation media presently in use. Fifty one new media were designed for the selective isolation of eleven species groups of Nocardia, Rhodococcus and a-wcIV actinomycetes. The selectivity of these media was first assessed by comparing the growth of a range of cluster representatives on these new media and on a control medium (currently used for the isolation of the particular group being tested). Of these fifty one new media, eight were subsequently found to be sufficiently selective for their target clusters. These were then evaluated quantitatively and the most practically selective media will be used in soil isolation studies. These new media will be applied to a variety of soils from diverse habitats with the objectives of isolating unexploited actinomycetes with industrial potential by providing better quality biological material for screening.

Isolation and Identification Of Cellulose- and Lignin Degrading Actinomycetes From the Gut of Higher Termites (Termitidae)

Pasti, M-B^2, M-Basagia^1, G-Concheri^1, S.Cardinali^3, D.L.Crawford^2 and M.P.Nuti^1

^1 Dipartirnento di Biotecnologie Agrafie, The University, Padova, Italy,
^2 Department of Bacteriology and Biochemistry, University of Idaho, Moscow, USA and
^3 Cattedra di Micologia, The University, Udine, Italy

From the gut of termite workers (Macrotermes, Armitermes, Microcerotermes, Odontotermes) it was possible to isolate lignocellulosolytic actinomycetes by using selective conditions.

These consisted mainly of enrichment cultures in liquid media with cellulose as the sole carbon source followed by inoculation onto the surface of cellulose filters laid on solid media containing mineral salts. Alternatively inoculation was carried out on bilayered solid media plus Avicel as a carbon source.

The temperature for optimal growth of the isolates ranged from 28 C to 46 C; optimal pH for growth was 6.0 to 7.0. All isolates (20/20) were able to grow on pure cellulose, supplied either as filter paper or a microcristalline substrate (Pasti and Belli, FEMS Microbiol. Lett., 26, 107, 1985), and in the presence of 6.0xlO^-3 mM phenol or 1 mM guaiacol. Although all of them are able to grow on Kraft lignin in the presence of other carbon sources, only three isolates exhibited a strong ability to metabolize the former substrate.

The ability of the isolates to degrade lignin was assessed by monitoring the degradation of labelled substrates and the consequent formation of C, determining substrate losses during growth on purified lignocellulose, and quantifying the amount of APPL (acid precipitable polymeric lignin) formed during substrate degradation (Pasti et al., paper submitted, 1990).

Most isolates produce extracellular peroxidases and esterases, some of which are inducible in the presence of wheat straw. Twelve strains were identified using the probabilistic matrix of Williams et al. (J. gen. Microbiol., 129, 1815, 1983) and the matrix of Langham et al. (J. gen. Microbiol., 135, 121, 1989). The identified strains were found to belong to the clusters Streptomyces chromofuscus, S. rochei and S.diastaticus.

New Approaches to the Identification of Streptomycetes Which Produce Novel Bioactive Compounds

Phillips, L., E-M.H.Wellington and S.Rees*

Department of Biological Sciences, University of Warwick, Coventry and *Microbiology Section, ICI Agrochemicals Division, Bracknell, Berkshire, UK

As the number of known antibiotics increases it becomes more difficult for industry to discover new bioactive compounds. Methodologies which enhance the discovery of new antibiotics and improve the identification of those organisms capable of producing them are required to counteract this problem. There are many innovations within our group which aspire to improve the identification of bioactive strains.

Certain physiological characters may be diagnostic for bioactivity, current research involves investigating antibiotic resistance, fatty acid and metabolite profiles as phenotypic markers for predicting bioactivity. The bioactivities of streptomycete isolates are superimposed onto their phenotypic distributions. Progress is being made to show that correlations do indeed exist between these two variables.

Systems for wide gene expression are under development, where strains are exposed to wide ranging environmental conditions, using minimal amounts of time and resource. We also hope to investigate the specificity and application of certain physiological factors to the provocation of wide gene expression and therefore gain better understanding of environmental factors affecting production.

Removing the ability to produce known broad spectrum compounds may allow isolates to produce new compounds or facilitate the detection of novel narrow spectrum compounds. We are using a model system to investigate these effects and hope to develop repression systems for a particular polyether and ansamysin antibiotics.

Selected Problems and Approaches to Actinomycete Taxonomy

Prauser, H.

Zentralinstitut fur Mikrobiologie und Experimentelle Therapie, Jena, DRG

Actinomycete taxonomy, dominated for decades by the use of morphological, physiological and ecological characters and culminating in their statistical processing, entered the molecular level with the elucidation of peptidoglycan structures. These and further chemotaxonomic characters were understood to reflect phylogenetic relationships more closely than the attributes used before. Continuing this line, the base sequences (or their reflections, respective]y) of DNA, RNA and particularly 16S rRNA were used to achieve phylogenetic evidence. Both in phenetical and phylogenetical studies the comparisons of the organisms or the macromolecules data sets are presented in the form of trees or other graphical approaches. The trees need to be translated into hierarchical systems as bases of worldwide communication on the microorganisms being and capacities. This means, the levels of ramifications must be defined to determine the nomenclatural ranks, particularly those of the species and the genus. As a matter of principle and of experience, this can only be performed empirically. i.e., the phylogenetic relationships of the organisms will be elucidated more and more in detail, and the systems to be used in biological sciences and in practice will be continuously adopted; but they will always be man-made and, consequently, a subject of controversy. Among the problematic examples are the classificatory situations of Promicromonospora, Nocardioides/Pimelobacter, Nocardia/Rhodococcus and of the streptomycetes.

The standards concerning the description of new actinomycete genera need to be reconsidered. Circumscriptions should be wide enough to diminish the necessity of future emendations. Characters of doubtful discriminating value may be: motility, formation of hyphae and branches, presence and definition of spores and sporangia, number of spores in chains, relation to oxygen, fatty acid patterns and others. The growth cycle including the occurring morphological structures should be given and documented by micrographs. Basic chemotaxonomic characters are essential.

Identification of strains at the genus level is most easily and rapidly performed by phage typing with specific phages. Now, they are available for most of the nocardioforms and for a part of the sporoactinomycetes. Attempts at isolating phages for coryneforms turned out to be less promising.

An undefinable fraction of existing microorganisms has apparently not been detected as yet. The evidencing of Agromyces ramosus in soils and of nocardioforms in gut and feces of invertebrates indicates future ways of discovering unknown actinomycetes. Nocardioideae, Promicromonosporae and Oerskoviae, growing readily on ordinary media, predominate in nearly pure mineral substrates. In contrast to their phages, they were only rarely isolated from soils. Oerskoviae could be likewise demonstrated in clinical specimens as opportunistic pathogens. The question is obviously an ecophysiological puzzle.

Selective Isolation of Thermoactinomycetes in the Absence of Antibiotics

Priest, F.G. and E.F.McPherson*

Department of Biological Sciences, Heriot-Watt University, Edinburgh and *Department of Microbiology, The University, Aberdeen, UK

Selective isolation of thermoactinomycetes relies on the natural resistance of these bacteria to novobiocin. This is very effective but makes no allowance for taxa which might be sensitive to the antibiotic. Indeed, the atypical mesophile, T. peptonophilus does not display novobiocin resistance.

We have developed a procedure for selective isolation of thermoactinomycetes which is based on differential inhibition of spore germination by L-serine. In brief, heat-treated samples are cultured in the presence of L-serine. Thermophilic Bacillus spores germinate but those of thermoactinomycetes do not. A subsequent heat-treatment kills the thermophilic bacilli and the thermoactinomycete spores are then allowed to develop into colonies. We have used this procedure to isolate thermoactinomycetes from a variety of habitats.

The Effect of Agitation Rate on Pellet Growth of Streptomycetes

Prosser, J.I. and A.J.Tough,

Department of Molecular and Cell Biology, The University, Marischal College, Aberdeen, UK

A model is presented for the development of pelleted populations of filamentous microorganisms in submerged culture. Predictions of the model have been tested experimentally using Streptomyces coelicolor A3 (2) grown in semi-defined medium.

The mathematical model considers formation and growth of pellets from a spore inoculum. The radial growth rate of pellets is assumed to be constant in batch culture and proportional to substrate concentration in continuous cultures. Radial growth results from biosynthesis in an outer mycelial shell, the peripheral growth zone, within which mycelial density increases with increasing distance from pellet margin. New pellets are formed by fragmentation and break-up of existing pellets at a rate dependent on shear forces and agitation rate. The model generates predictions in the form of pellet size distributions, in addition to total biomass concentration, pellet number and substrate concentration. These predictions suggest an inverse relationship between pellet radius and agitation rate and a reduction in the critical dilution rate with decreasing agitation rate. Decreasing dilution rate at constant agitation rate has the opposite effect, decreasing pellet radius and increasing pellet number.

Predicted effects of agitation rate have been tested in batch and glucoselimited continuous cultures of S. coelicolor. In the former, agitation rate influenced pellet size distributions by affecting the aggregation of spores prior to pellet formation. Growth was complete, however, before agitation could significantly influence size distributions through effects on fragmentation processes. In continuous culture, experimental results and model predictions showed good qualitative agreement but quantitative discrepancies arose through difficulties in defining accurately the resistance of pellets to shear forces and consequent break-up. The model therefore provides a valuable mechanistic basis for pellet growth kinetics and highlights a major deficiency in our knowledge of mycelial growth.

Isolation and Characterisation of Alkalophilic Actinomycetes

Saddler, G.S. and M-Goodfellow*

Lepetit Research Center, Gerenzano, Italy and *Department of Microbiology, The University, Newcastle upon Tyne, UK

Large numbers of alkalophilic actinomycetes were isolated from a variety of soils using a number of different isolation media adjusted to pH 10.0. An artificial classification of 731 alkalophillc isolates based on pH requirements for growth, morphology and pigmentation properties revealed that 80% of these strains were able to grow at pH 10.0 and pH 7.0 and that they were recovered in 25 colour groups. These strains which were considered alkalotolerant possessed morphological characters consistent with their inclusion in the genus Streptomyces. One hundred and seventy representatives of the colour groups were compared with marker strains of established Streptomyces species for 136 unit characters. Data were examined using standard numerical taxonomic procedures. Results show that the majority of alkalotolerant isolates were distinct from the Streptomyces marker strains.

Identification of Streptomycetes From Grapevine Carposphere

Vercesi, A., E.Volpi and R.Locci*

Institute of Plant Pathology, The University, Milan and *Chair of Mycology, The University, Udine, Italy

Two-hundred and twenty streptomycetes isolated from grapevine carposphere were identified using the probability matrix of Williams et al. (J. gen. Microbiol., 129, 1815, 1983). Just 17% of the isolates could not be identified with any of the clusters of the matrix.

Most of the identified strains were placed in the cluster Streptomyces albidoflavus and the remaining ones in the clusters albus, atroolivaceus, chromofuscus, cyaneus, diastaticus, exfoliatus, griseoruber, phaeochromogenes and rochei.

Forty-eight per cent of the strains show antibiotic activity against 107 yeasts isolated from the same habitat and associated with grapevine sour rot. Most antagonistic strains belong to the albidoflavus phenon. Lower activity is shown by representatives of the other clusters.

The Development of Identification Methods for Actinomycetes Based Upon the Polymerase Chain Reaction

Warwick, S., J. Challans, T.Bowen and M.Embley

Microbial Technology Research Unit, Polytechnic of East London, London, UK

Oligonucleotide probes directed against the variable regions of 16S ribosomal RNA (rRNA) have proved useful in the identification of many bacteria including actinomycetes. However, it is sometimes difficult to extract sufficient RNA from actinomycetes to probe and to carry out the comprehensive evaluation of putative probes. We have used a rapid guanidium isothiocyanate - based method for the extraction of DNA from single colonies. Primers have been designed to specifically amplify 16S rRNA genes using the polymerase chain reaction. The technique is easy and rapid and gives sufficient product for many probing experiments. The technique has been extended to an asymmetric amplification system which allows direct sequencing of the amplified rRNA genes. The sequences obtained can be used to give a phylogenetic identification of the organism being investigated.

Immunoblot Analysis of Biotin-containing Proteins in Nocardia Ssp. And Rhodococcus Ssp.

Well, H.-P. and K.P.Schaal

Institut fur Medizinische Mikrobiologie und Immunologie der Universitat, Bonn, FRG

Analyzing bacterial cytoplasmic proteins by polyacrylamide gel electrophoresis (PAGE) may be a useful approach for the identification of bacterial strains. However, this method is hampered by the number of proteins present and their variability due to culture conditions and preparation methods. Only direct comparison of such preparations may help to determine the identity of an unknown bacterial strain. We tried to reduce the heterogeneity of such a protein pattern by selecting only biotin-containing proteins as markers of bacterial strains. Therefore after PAGE the proteins were blotted onto nitrocellulose membrane. As a probe we used the protein streptavidin from Streptomyces avidinii which has a high affinity to biotin. Streptavidin was conjugated to horseradish peroxidase and the binding to the biotin- containing proteins was visualized by the enzymatic activity on the nitrocellulose blots. Nocardia asteroides, Nocardia farcinica, Nocardia brasiliensis and Nocardia otitidiscaviarum were analyzed by this method. Within a species different strains expressed the same biotinylated proteins. Comparing different species five major proteins were present in all preparations: p50, p64, p75, p87, and p99. N. farcinica and N. brasiliensis had an additional p150 which was not found in N. asteroides and N. otitidiscaviarum. The latter species was characterized by a protein (p60) which was not detectable in the other species tested. Preliminary results with Rhodococcus ssp. indicate that the five nocardial major proteins are only expressed in some, but not in all Rhodococcus ssp. analyzed, and additional biotinylated proteins do occur. We conclude from these results that analyzing biotin-containing proteins may be a helpful tool to differentiate Nocardia ssp. and Rhodococcus ssp.

New Methods for the Detection and Recovery of Streptomycetes in Soil.

Wellington, E.M.H, D.McDowell, N. Cresswell, P.R.Herron P.W.Baker, A.Wipat* and V.A.Sunders*

Department of Biological Sciences, University of Warwick, Coventry and *School of Natural Sciences, Liverpool Polytechnic, Liverpool, UK

The distribution and diversity of streptomycetes in soils is unknown. A limited number of studies have attempted to characterize isolates obtained from various selective media. However, isolation techniques such as dilution plating are mostly selective for spores. It is possible that spore inoculum is so widely dispersed that little difference in diversity is found between differing soil types. We are trying to develop methods to determine the active components of streptomycete populations in soil and also to assist in survival studies for monitoring activity of genetically engineered strains (GEMs) in soil. The use of soil microcosms has enabled detailed studies of the growth and survival of both wild types and GEMs in soil. We have been able to investigate the life cycle of various strains in soil and determine the recovery of sporulating and non-sporulating inoculants using spore specific extraction procedures and scanning electron microscopy. Alternative methods for detection and quantification of streptomycetes have been developed using nucleic acids extracted directly from soil. DNA and RNA isolated can be probed for specific sequences and enable detection of both phage and plasmid DNA. The usefulness of this approach relates to sensitivity which is dependent on the relative efficiencies of extraction. Analysis of DNA isolated from soil may help to provide data on species diversity.

Application of High Performance Liquid Chromatography for Determination of Dap (Diaminopimelic Acid) in Cell Walls of Actinomycetes

Wolstrup, J. and N.O.G-Jorgensen,

Department of Microbiology, R.Vet. and Agricultural University, Frederiksberg, Denmark

Quantitative determination of cell wall components, including diaminopimelic acid (DAP), is essential in identification of actinomycetes.

The purpose of the present work was to apply a rapid HPLC (high performance liquid chromatographic) method for analysis of amino acids to separate and identify LL-DAP and meso-DAP in actinomycetes. A more accurate determination of DAP is required, especially for actinomycetes containing both isomers of DAP, such as Kitasatosporia. In the present HPLC method DAP was measured as fluorescent o-phthaldialdehyde derivatives according to Lindroth & Mopper (Anal. Chem., 52, 1667, 1979) and Jorgensen (Limnol. Oceanogr., 32, 97, 1987). In addition to being rapid, this method allows analysis of very small amounts of sample. Thus, in contrast to analyses by paper chromatography, where relatively large amounts of biomass are needed, actinomycete cells harvested directly from a petri dish produce sufficient material for analysis.

The harvested biomass was mixed with 100 ul 6M HCl in a closed centrifuge glass and hydrolyzed at 100 C for 18h (Komagata and Suzuki, In: Methods in Microbiology. Academic Press, London, 19, 1987). After filtration through a 0.45 um membrane filter the sample was diluted to 10 ml with Milli-Q water (Millipore, Ass.). An additional dilution of 1:3 was normally required before a 150 ul sample was injected into the HPLC. 20 different strains of actinomycetes from Novo-Nordic, Denmark, were analyzed in this study.

Copyright 1990 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia