Lysozyme Sensitivity in Streptoverticillium and Streptomyces Species

System. Appl. Microbiol. 6, 239-242 (1985)

Lysozyme Sensitivity in Streptoverticillium and Streptomyces Species RICHARD W. ATTWELL 1, ALLAN SURREY\ and TOM CROSS 2 1

Department of Biological Sciences, Manchester Polytechnic, Manchester M1 5GD and 2Postgraduate School of Biological Sciences, University of Bradford, Bradford BD7 1DP, England

Received January 22, 1985

Summary Representative strains from the major taxonomic clusters within the genus Streptoverticillium were able to grow in the presence of lysozyme. The Streptomyces strains examined were sensitive to the enzyme. The only exceptions were strains of the Streptomyces rimosus and Streptomyces lavendulae clusters. Mycobacterium phlei and Micromonospora chalcea strains were lysozyme sensitive; results which conflict with some previous reports but which probably reflect differences in test systems. Growth of the resistant streptoverticillia and streptomycetes caused a decrease in lysozyme activity in the sensitivity test medium. The results indicate that the enzyme was not inactivated by a soluble inhibitor or protease but was bound to the mycelium and removed from the medium. The findings are discussed in the context of streptomycete classification and of the development of a selective isolation method for Streptoverticillium species.

Key words: Lysozyme Sensitivity - Streptoverticillium - Streptomyces - Taxonomy - Isolation methods

Introduction The majority of strains used in taxonomic and other studies on the genus Streptoverticillium (Cross et aI., 1973; Konev and Mitnitskii, 1980; Locei et al., 1981) were isolated originally in screening programmes for biologically active compounds. This may have given a biased view of the genus and a greater variety of strains from natural substrates are needed to confirm and extend our knowledge of this taxon. Many Streptoverticillium species synthesise biologically active secondary metabolites, some of which have been produced commercially. But because strains are isolated infrequently the genus has not been subjected to the same intensive examination and industrial exploitation as the closely related genus Streptomyces. There is a need for an efficient isolation system to provide a wide range of Streptoverticillium strains for systematic studies and to screen for useful products. Such a system must include means for decreasing competition from other bacteria, particularly the streptomycetes which grow well on media suitable for streptoverticillia. The lysozyme resistance of representatives of certain Streptoverticillium species reported by Kutzner et al. (1978) could be a useful selective characteristic. Kutzner et al. (1978) used lysozyme at a final concentration of 100 !-tglml but did not specify enzyme activity or test condi16

System. App!. Microbia!. Va!. 6

tions. The action of lysozyme on actinomycetes in general has been studied by a variety of test methods. In the context of an isolation system it is important to assess the effect of the enzyme on bacterial growth (Gordon and Barnett, 1977) rather than on isolated wall material (Kawamoto et al., 1981) or non-growing biomass (Mordarska et al., 1978). This paper describes the effect of lysozyme on the growth of strains of the genera Streptoverticillium and Streptomyces. Strains representing major clusters reported in recent taxonomic studies were used (Locci et al., 1981; Williams et al., 1983).

Materials and Methods Bacteria and culture conditions The Bacteria studied are listed in Table 1. Strains of the species

Arthrobacter citreus, Arthrobacter globiformis, Micromonospora chalcea and Mycobacterium phlei were included as controls and to compare the results of the study with those reported by other workers (Gordon and Barnett, 1977; Kawamoto et al., 1981). All bacteria were cultured on YME agar (ISP2, Shirling and Gottlieb, 1966) incubated at 28°C for 7 days. Spores, or vegetative growth in the case of Mycobacterium and Arthrobacter spp, were suspended in sterile saline for use as the inoculum in subsequent tests.

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R. W. Attwell, A. Surrey, and T. Cross

Lysozyme senstivity tests

Results and Discussion

Sensitivity tests were carried out in shake flasks (100 ml) containing 50 ml of tryptone-yeast extract broth (ISP1, Shirling and Gottlieb, 1966) and lysozyme (Sigma Chemicals, St Louis) to give an activity of 468 units/ml. The lysozyme concentration chosen was within the range used by Gordon and Barnett (1977). Control cultures were set up in the same medium without added lysozyme. Inoculated flasks were incubated at 28°C on an orbital shaker at 180 rpm. Results were recorded after 7 days. Lysozyme activity was determined at 0, 2 and 7 days during sensitivity tests on strains of Streptoverticillium baldaccii, Stv. griseoverticillatum, Streptomyces aurantiacus and Stmy, kanamyceticus. Samples were centrifuged and the lysozyme activity in the supernatant was determined by means of a standard bioassay employing Micrococcus luteus. Fresh lysozyme was added to the supernatant obtained from 7 days sensitivity test cultures (ie with lysozyme) of Stv. griseoverticillatum and Stmy. kanamyceticus to give a final concentration of 468 units/ml. Activity was determined after the solution had been held at 28°C for 30 min. Biomass, obtained from centrifuged 7 day old control cultures (ie without lysozyme) of Stv. griseoverticillatum and Stmy. kanamyceticus, was resuspended in 50 ml of fresh tryptone-yeast extract broth containing 468 units/ml of lysozyme. Enzyme activity in the medium was determined after the resuspended culture had been shaken at 28°C for 30 min.

Streptoverticillium strains representing the major clusters proposed by Locci et al. (1981) were able to grow in the presence of lysozyme (Table 1). This confirms and extends the results obtained by Kutzner et al. (1978) using a different test system. The majority of the Streptomyces strains tested did not grow in the presence of lysozyme. The exceptions were strains of species in the Stmy. rimosus cluster group B-42 (Williams et al., 1983) and in the Stmy. lavendulae cluster group F-61 (Williams et al., 1983). The latter observation is particularly significant because the species Stmy. lavendulae shows a number of similarities with the streptoverticillia (Silvestri et al., 1962; Williams et al., 1983). The difference in lysozyme sensitivity between those members of the two closely related genera examined could be a useful character in future taxonomic studies. It could be exploited also in an isolation system selective for streptoverticillia to reduce interference from associated streptomycetes. The four control bacteria were all found to be lysozyme sensitive (Table 1). This agrees with results reported by Gordon and Barnett (1977) for Mycobacterium phlei

Table 1. Lysozyme sensitivity in Streptoverticillium and Streptomyces species

Streptoverticillium spp

baldaceii biverticillatum griseoverticillatum cinnamoneum luteoverticillatum hachijoense aureoversales ladakanum mobaraense aspergilloides ehimenuse olivoreticuli orinoci

ATCC 23654 ATCC 23615 ATCC 27436 ATCC 11874 ATCC 23933 ATCC 19769 IPV 2051 ATCC 27441 ATCC 29032 ATCC 14808 IPV 2016 IPV 2252 ATCC 23202

Cluster Sensitivity (Locei et al., 1981)

Streptomyces spp

1 1 1 2 3 4 5 6 7 8 9 10

albidoflavus griseus diastaticus coelicolor erythraeus ganmycius rimosus albidofaciens kanamyceticus griseoluteus aurantiacus lavendulae lavendulocolor fradiae roseolilacinus

a

R R R R R R R R R R R R R

Additional control species:

Cluster

Sensitivity

(Williams et aI., 1983)

Micromonospora chalcea Mycobacterium phlei Arthobacter citreus Arthobacter globiformis

R S

Resistant: growth equivalent to that in the lysozyme free control. Sensitive: no growth in test system, growth in lysozyme control.

a ATCC IPV

Slight growth in test system after 7 days not included in a major cluster American Type Culture Collection Istituto di Patologia Vegetale (Milan)

ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC

25422 23345 3315 23899 11635 27434 10970 25184 12853 12768 19822 8664 15871 10745 19922

A A A A A A B B B C C F F

G G

S S S S S S R R R S S R S,· S S S S S S

Lysozyme Sensitivity in Streptoverticillium and Streptomyces Table 2. Decrease in lysozyme activity after 2 and 7 days incubation in sensitivity test. Initial lysozyme concentration was 468 units/ml

Lysozyme Sensitivity

Organism

Streptoverticillium baldaccii Stv. griseoverticillatum Streptomyces aurantiacus Stmy. kanamyceticus Uninoculated control R = Lysozyme resistant

ATCC ATCC ATCC ATCC

23654 27436 19822 12853

R R S R

S = Lysozyme sensitive

where a growth challenge type test was also used. Other workers, (Sohler et aI., 1958; Mordarska et aI., 1978; Kawamoto et aI., 1981) have reported Mycobacterium phlei and Micromonospora chalcea to be lysozyme resistant but the test was carried out on non-growing biomass or on isolated cell walls. Such methods determine enzyme action on mature wall material. Tests performed on growing cells also take account of lysozyme action on the growing hyphal apex, a small but important part of the mycelial biomass, which may differ in wall structure from older regions ~of the hyphae (Locci, 1980). The results of tests which monitor the action of lysozyme on growing cultures are more useful for the development of selective media. Lysozyme activity was monitored in sensitivity test systems for two Streptoverticillium strains and for one resistant and one sensitive streptomycete strain (Table 2). Activity fell by more than 50 % after 7 days in uninoculated medium and also in the flasks inoculated with the sensitive organism in which no growth occurred. In the supernatant from growing resistant cultures no activity could be detected after 7 days. Two possible mechanisms were considered for the noted decrease in lysozyme activity brought about by the resistant bacteria. First, that lysozyme was inactivated by an extracellular protease or an enzyme inhibitor produced by the culture. Second, that the lysozyme became associated with the growing biomass and was thus removed from the test medium. Lysozyme added to the supernatant after removal, by centrifugation, of the mycelium of the resistant strains, Stv. griseoverticillatum ATCC 27436 and Stmy. kanamyceticus ATCC 12853, remained fully active for 30 min at 28°C. When the mycelium of these resistant strains was resuspended in fresh medium containing lysozyme there was a rapid loss of activity in the liquid phase in the same time period; 76 % loss in the case of Stv. griseoverticillatum and 100 % loss for Stmy. kanamyceticus. The results indicate that the enzyme was not inactivated by extracellular enzymes or inhibitors. The lysozyme became associated with the mycelium and was either inactivated by surface enzymes or adsorbed to the wall which must have retained its structural rigidity to prevent lysis during subsequent cell growth. The activity or fate of the adsorbed lysozyme has yet to be determined. Members of the genera Streptoverticillium and Streptomyces share high DNA homology values (Okanishi et aI., 1972) and are similar in other respects including wall chemotype (Lechevalier and Lechevalier, 1970), G + C

241

% decrease in lysozyme activity

2 days

56 54

48 100 ND

7 days

100 100 59 100 51

ND = Not determined

content of their DNA (Pridham and Tresner, 1974), menaquinone composition (Minnikin and Goodfellow, 1981; Goodfellow and Cross, 1984) and phage sensitivity (Wellington and Williams, 1981). Particular aspects of wall composition in strains of both genera are therefore under investigation to find the basis for their different responses to lysozyme. In addition, work is in progres on the development of a selective medium, incorporating lysozyme, for the isolation of streptoverticillia.

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Dr. R. W. Attwell, Dept. of Biological Sciences, Manchester Polytechnic, Manchester M1 5GD, England