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Hartmannella

Acanthamoeba

Oda

Hsa Peter H.H. Weekers

Acanthamoeba and Hartmannella. Ecophysiological, biochemical and molecular biological differences Cover: Isoenzyme patterns, a phylogenetic tree, and bacteria; Acanthamoeba castellami and Hartmannella vermiformis in their context (photographs by Dr. CK. Stumm). Acanthamoeba and Hartmannella. Ecophysiological, biochemical and molecular biological differences

EEN WETENSCHAPPELIJKE PROEVE OP HET GEBIED VAN DE NATUURWETENSCHAPPEN

PROEFSCHRIFT

TER VERKRIJGING VAN DE GRAAD VAN DOCTOR AAN DE KATHOLIEKE UNIVERSITEIT NIJMEGEN, VOLGENS BESLUIT VAN HET COLLEGE VAN DECANEN IN HET OPENBAAR TE VERDEDIGEN OP WOENSDAG 30 JUNI 1993, DES NAMIDDAGS TE 3.30 UUR PRECIES

DOOR

PETRUS HENRICUS HUBERTUS WEEKERS

GEBOREN OP 2 JUNI 1959 TE WEERT Promotor: Prof. Dr. Ir. G. D. Vogels

ISBN: 90-9006142-8

The studies described in this thesis were financially supported by The Netherlands Integrated Program for Soil Research (PCBB), The Netherlands Organisation for Scientific Research (NWO) and the National Institute of Health (USA). The investigations described in this thesis were carried out at the Department of Microbiology, University of Nijmegen, The Netherlands (Chapter 2-5 and 7), at the Institute of Hygiene and Epidemiology, Brussels, Belgium (Chapter 6) and at the Department of Molecular Genetics, Ohio State University, Columbus, Ohio, USA (Chapter 8). Contents

Chapter 1. General introduction. 1

Chapter 2. Effects of grazing by the free-living soil amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis on various bacteria. 19

Chapter 3. Mass cultivation of the free-living soil , Acanthamoeba castellana in a laboratory fermentor. 31

Chapter 4. Axenic cultivation of the free-living soil amoebae, Acanthamoeba castellana and Hartmannella vermiformis in a chemostat. 45

Chapter 5. Nitrogen metabolizing enzyme activities in the free-living soil amoebae, Acanthamoeba castellana, Acanthamoeba polyphaga and Hartmannella vermiformis. 55

Chapter 6. Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella. 67

Chapter 7. Bacteriolytic activities of the free-living soil amoebae, Acanthamoeba castellana, Acanthamoeba polyphaga and Hartmannella vermiformis. 81

Chapter 8. Small-subunit ribosomal RNA analysis of three Hartmannella vermiformis strains. Sequences, secondary structure and phylogenetic implications. 95

Summary/Samenvatting 111

Dankwoord 117

Curriculum vitae 119

Publications 121 aan mijn ouders Chapter 1

General introduction

Historical perspective

The word amoeba (Greek: αμοιβή) means change and suggests a continuous metamorphosis. Antoni van Leeuwenhoek (1632-1723) probably discovered the first protozoan when he described a Vorticella-Ше organism in 1674. Although recognized as the putative "father of protozoology", he probably never discovered any organisms like amoebae [55, 79]. Until more than a century after the invention of the microscope Rosei von Rosenhof described in 1755 a microorganism which he called "little Proteus" [47, 55, 79]. Almost immediately after the discovery of "little Proteus" the uncertainty about this creature's name began and several different names, like Volvox , Chaos proteus and Proteus diffluens, were given to the same organism [55]. After the discovery of the amoebae only little progress was made in the 18* century, most of the amoebae were described in the 19,h century. In particular between 1830 and 1840 Dujardin in France and Ehrenberg and von Siebold in Germany helped to advance our understanding of the amoebae [47, 55]. The efforts of the IS"1 and 19th century scientists were directed mainly towards a description of more and more organisms and their classification, which became gradually easier as the quality of the available microscopes improved. Approximately 200 amoebal species were estimated by Schaeffer in the beginning of the 20* century [76]. More recent guides to freshwater and soil amoebae list over one hundred species, excluding the parasitic and testate representatives [68, 82], and an additional 40 species from marine habitats were described by Page [67]. In the first half of the 20* century these earlier, morphologically based, descriptions were further extended and became more accurate. However, since a few decades amoebae seem to become more important for scientists especially because the interests in soil-microbial ecology [18, 37, 41, 45, 83, 84], cell biology [13, 15, 17, 49], molecular biology [14, 16, 46] and especially phytogeny [9, 22, 50, 51, 77, 86].

1 Classification and phylogeny of amoebae

Traditionally, the amoebae were classified based upon morphological characteristics and two major groups were distinguished, those with lobose (= relative broad) pseudopodia and those with filose (= thin thread-like) pseudopodia. and identification of representatives of the various taxa (orders, suborders, families) depend on size, type of locomotion, locomotor organelles, patterns of nuclear division, presence or absence of a test or surface scales, cyst formation and shape, and the type of habitat. A tentative classification of amoebae with lobose pseudopodia is shown in Table 1. At the present time still much disagreement exists about taxonomy, systematics and origins of amoebae based upon the traditional morphologically based classification [11, 23, 54, 66, 68, 69, 79, 82, 85]. Progress in taxonomy and phylogeny of amoebae is slow. The use of modern methods like biochemical and physiological analyses has only just begun and serological and immunological relationships have only been established recently [24, 25, 31, 32, 61, 71, 96]. Also molecular biological methods, especially small-subunit ribosomal RNA (srRNA) analysis, have only recently been applied [9, 22, 46, 50, 51, 77, 86, 87]. Interpretation of the most recent data concerning the phylogeny of the amoebae leads to the conclusion that the group is probably polyphyletic [9, 11, 20, 22, 68, 86]. Phylogenese relationships among organisms (Fig. 1) show the diversity of the eukaryotic protozoa as a group with Naeglena and Acanthamoeba as the free-living amoebal representatives. Schuster [78] indicates that amoebae probably originated from mastigote ancestors through the loss of kinetids because a close link between amoebae and mastigotes can be found by comparison of the amoebae stage of Naeglena and Tetramitus with rhizopods such as Vahlkamphia and Hartmannella. A tentative scheme for the origin of amoebae has been suggested by Bovee and Jahn [11] in which e.g. both Acanthamoeba and Hartmannella descend from the pre-acrasids. However, although recently collected morphological, physiological, biochemical and molecular biological data will refine the taxonomie relationships among amoebae, their phylogeny is likely to remain disputable.

2 Table 1. Taxonomy of amoebae with lobose pseudopodia. Adapted after Page [68], Sleigh [85] and Schuster [79].

Subclass Gymnamoebia Naked

Order Euamoebida Family Amoeba, Chaos. , Tnchamoeba Family Hartmannella, Saccamoeba, Cashia Family Thecamoeba, Pseudothecamoeba Family , Ρ la tyamoeba Family , , Dactylamoeba Family Vexilhfendae Family Vahlkamphidae8 Vahlkamphia, Naeglena, Tetramitus Family , Endohmax

Order Acanthopodida Family Acanthamoeba, Protacanthamoeba Family Echinamoebidae Echmamoeba, Filamoeba

Order Family Leptomyxidae Leptomyxa Family Flabellulidae , Parqflabellula

Subclass Testacealobosia Tests or shell enclosing body.

Order Arcellmida Family Arcellidae Arce Ila Family Centropyxìs Family Difflugndae

Order Trichosida Tnchosphaenum

8 according to Page [68], Sleigh [85] and Page & Siemensma [69] classified in the Class Heterolobosea but according to Schuster [79] in the Class Lobosea according to Schuster [79] classified in the Class Lobosea but according to Page [68] and Page & Siemensma [69] having no ordinal relationship

3 EUKARYA

+ *** ^reu 9

. Plasmodium falaparum Dictyostelium discoideum *^>я> — Entamoeba histolytica Naeglena gruben Euglena gracilis Trypanosoma brucei Leishmania donovanii

Vainmorpha necatrix Giardia lamblia

Halobactenum volcanii Sullolobus solfatancus

ARCHEA

Fig. 1. Phylogenetic tree based on small-subunit ribosomal RNA analysis showing the eubacteria (BACTERIA), archaebacteria (ARCHEA) and (EUKARYA). The tree was derived using distance matrix methods in which the line segment lenghts represent the evolutionary distance between the organisms Modified after Sogin [86].

Amoebal cells, diversity and multiplication

Amoebae are single-celled organisms ranging in size between a few μπι and a few mm, either naked or having a test and motile by means of pseudopodia, which are also used for phagotrophy. Among amoebae there is a widespread lack of recognized sexuality [79]. The life cycle of mostfree-living amoeba e is simple. The active cell or feeding stage in which multiplication takes place, is called amoeba or trophozoite and reproduces by equal binary fission. The inactive or dormant cell, called the cyst, exists for many though not all amoebae and is formed to survive unsuitable situations. Generation times are

4 highly variable among amoebae and generally increase with temperature and decrease with body size. However, even for amoebae of the same size growing at the same temperature, maximum rates of cell division vary widely among taxa [89].

Fig. 2. Diversity among the amoebae. (A) Vahlkamphia avaria, (В) Naegleria, (С) Stachyamoeba lipophora, (D) Thecamoeba sphaeronucleolus, (E) Vannella miroides, (?) Amoeba proteus, (G) Mayorella bigemma, (H) Vexillifera telemathalassa, (I) Hartmannella vermiformis, (J) Chaos illinoisense, (K) Saccamoeba lucens, (L) cloaca, (M) Echinamoeba exudans, (N) Acanthamoeba, (O) Filamoeba nolandi, (P) Hylodiscus rubicundus. Scale = 10 μτα for A, B, C, E, I, M; 15 цт for H, N, O, P; 30 цт for D, G, K, L; 50 μηι for F; 100 цт for J. After: Taylor and Sanders [89] (published with permission).

5 The primary characteristic of amoebae is the possession of some forms of pseudopodia which show considerable diversity of structures and types, reflected in the variety of shapes which consists among the free-living naked amoebae (Fig. 2). The larger amoebae (e.g. Amoeba proteus) have long served as a model of a single-cell system in introductory biology courses [4] and for studying amoeboid locomotion [12]. Only recently relatively smaller amoebae, and especially Acanlhamoeba, have gained in popularity for cell biological studies [13, 15-17, 49] and for the study of nonmuscule contractile systems [3, 12, 72]. The smaller amoebae (< 20 μπι), which occur in water and soils, are more difficult to detect because of their size. They also lack clear morphological features, and can only be cultivated by means of specialized methods [68]. The cell body organisation of amoebae has a subcellular nature and the diversity of anatomical features as seen in the "higher eukaryotes" is absent. Two zones can be distinguished in the cytoplasm of amoebae. A thin transparent outer layer or ectoplasm and a more granular endoplasm which forms the bulk of the cytoplasm and which flows forward through the body into the cylindrical pseudopodia (Fig. 3).

Fig. 3. Schematic drawing of an amoeba cell, (cv) contractile vacuole, (fv) food vacuole, (p) pseudopodia and (n) nucleus.

6 The ectoplasm is most clearly visible at the tip of a pseudopodium where it forms a hyaline cup. The endoplasm contains one or many nuclei, a variety of vacuoles, food storage granules, mitochondria and other membranous or crystalline inclusions [4].

Nutrition and cultivation

There are no such culturing conditions in which only amoebae are able to grow without supporting also growth of other types of protozoa. For their growth, heterotrophic amoebae require preformed compounds, either complex molecules or their simple breakdown products. These food compounds may be particulated, semi-solid or in solution and are absorbed by means of endocytosis [29, 52]. The phagotrophic lobose amoebae engulf microorganisms like other protozoa and bacteria which they encounter with their pseudopodia as they creep around, and which they use as a food source. Among the larger amoebae are carnivores e.g. Amoeba and Chaos, eating mainly ciliates and flagellates, and herbivores, e.g. , eating mainly diatoms, while the smaller forms, e.g. Acanthamoeba and Hartmannella, feed mainly on bacteria. Especially among these small amoebae food preferences for bacteria occur [37, 83, 84]. Once food capture is completed, the food vacuole separates from the surface plasma membrane but the food does not pass directly into the cytoplasm, the food vacuole keeps it separate from the cell constituents. The food passes through a series of changes, starting with death of prey due to dehydration and followed by its gradual digestion over several hours [12, 19, 29, 62]. Whether food uptake occurs by means of phagocytosis of particles or other organisms, or pinocytosis of molecules the process is essentially the same and accidental uptake of substances occurs during both processes [66]. For physiological and biochemical studies a mass cultivation technique for Amoeba proteus using axenic, washed Tetrahymena pyriformis as food source was developed by Prescott and James [73], and further perfected by Goldstein and Ko [42]. Yields (20-25 χ IO6 cells per ml) in such cultures were sufficient for biochemical characterization, organelle isolation, and other studies requiring large yields of organisms [43, 44]. Axenic cultures of carnivorous amoebae have not been established yet because these amoebae require food organisms

7 which they ingest. A more extensive methodology to establish axenic cultures in which amoebae use dissolved nutrients only, needs to be developed. Nutrition and growth of Acanthamoeba and other small amoebae was reviewed by Schuster [79] showing that only a few amoebae can be grown axenically. Among the free-living amoebae only Acanthamoeba and Naegleria grow readily in relatively simple media in the absence of bacteria [7, 30, 68, 80] and even in chemically defined media [1, 8, 81]. Both have already been used extensively for studies of encystment, phagocytosis and characterisation of extranuclear DNA [13, 15]. However, axenic cultivation of more amoebal species is needed, an undertaking that has not attracted many investigators yet.

Ecology

Amoebae show a cosmopolitan distribution in aquatic and terrestrial habitats and also occur as symbionts. The smaller soil amoebae are ubiquitous whereas the distribution of the larger amoebae depends on relatively restrictive physical and biological conditions [49, 55]. Numerous testate and naked amoebae are specialized for life in soil, where they form a significant component of the biota. Acanthamoeba and Hartmannella are very common soil and water amoebae and both species are very voracious bacteria-feeders although they may show some food preferences [37, 66, 83, 84]. Early in this century it was recognized that numerous active feeding protozoa exist in the water films around soil particles. Between a few thousand and half a million protozoa were estimated in each gram of moist soil, along with perhaps 109 bacteria on which they feed. Protozoa occur in considerably larger numbers in rhizosphere areas immediately around the plant roots than in the surrounding soil but both contain the same species [26-28]. Bacterivorous soil amoebae are smaller than aquatic amoebae and most have dormant cyst stages which permit them to survive periods of drought and limited food supply characteristic for soil related habitats. Small free-living amoebae are estimated to constitute 50-90% of heterotrophic protozoa in soils and litters [5, 6] and seem to be the most important bacterial predators in soil [2, 5, 6, 18, 21, 35, 36, 41, 48, 85, 88]. Most soil samples contain a wide variety of small bacterivorous amoebae which show distinct preferences for bacterial food. Not

8 all bacteria are equally well consumed and e.g. pigmented bacteria are avoided. Amoebae appear in their trophozoite stage if sufficient food bacteria are present in a moist environment, but will rapidly form cysts during periods of dryness and lack of food. Cyst formation in free-living soil amoebae is an important factor in allowing the organism to survive in a wide variety of environments.

Pathogenicity

Parasitic lobose and heterolobose amoebae are generally small (between 5 and 50 μιη) and are classified into various genera. The most important amoeba parasitic in man is Entamoeba histolytica, causing amoebic dysentery. Only axenic cultivation allowed the study of Entamoeba metabolism and mechanisms of pathogenicity [34]. Amoebae of several other genera are also common parasites of man and have disease potential, but most of these are non-pathogenic. Occasionally such soil amoebae as Acanthamoeba, Hartmannella and Naegleria are found in association with human diseases [33, 38, 56-58, 69, 91-93]. Several types of free-living amoebae, members of the genera Acanthamoeba and Naegleria are infectious agents [33, 57, 92, 93]. Both are known to cause insidious diseases in weakened people like infection of the central nervous system (primary amoebic meningoencephalitis and granulomatous amoebic encephalitis) as well as eye infections (amoebic keratitis) [10, 56, 65, 70], and are free-living opportunistic pathogens. Acanthamoeba and Naegleria have been recovered from freshwater, seawater, bottled mineral water, air, sewage, soil, compost, swimming pools, hydrotherapy baths, dental treatment units, dialysis units and contact lens cases, etc. [33]. The widespread presence of these opportunistic pathogens represents a threat to people in a compromised state of health [59, 79]. Acanthamoeba and Hartmannella were recently reported to be able to harbor Legionella pneumophila, a bacterium which causes Legionnaires disease and pathogenic to man [39, 40, 53, 60, 63, 74, 75, 90, 94, 95].

9 OUTLINE OF THE THESIS

The free-living amoebae of the genera Acanthamoeba and Hartmannella show a cosmopolitan distribution and occur in a wide variety of habitats. They are found in soil as voracious bacterial grazers thus enhancing the mineralization of nitrogen, but also occur as potential pathogenic symbionts in man and animal. Research on specific monoxenic amoebal-bacterial interactions and on cell physiology, biochemistry and molecular biology of the free-living amoebae is greatly enhanced if axenic cultures and large quantities of cells can be obtained. In the late finies and late seventies the first axenic cultures were established for Acanthamoeba [64] and Naegleria [30], respectively. Only recently some axenic cultures have been established for Hartmannella [40, 97]. This thesis compares representatives of the genera Acanthamoeba and Hartmannella revealing ecophysiological, biochemical and molecular biological differences between both genera, and also between individual species. Chapter 2 describes ecophysiological interactions between amoebae and bacteria in monoxenic cultures. Differences in food preference, growth yield and ammonium production for amoebae growing on various bacteria were determined. Chapters 3 and 4, both deal with growth and axenic cultivation of Acanthamoeba and Hartmannella. Mass cultivation of the free-living soil amoeba Acanthamoeba castellana was performed in laboratory fermentors in order to obtain gram quantities of axenic cells and to determine specific glucose and oxygen consumption, and ammonium production rates (Chapter 3). An easy and simple method for the axenic cultivation of Acanthamoeba and Hartmannella, yielding large quantities of cells was performed in chemostat cultures. Differences in generation time and specific respiration rates between representatives of both species were determined (Chapter 4). Chapter 5 describes the specific activities of some enzymes involved in the nitrogen metabolism of several species of Acanthamoeba and Hartmannella. Chapter 6 shows differences between axenically cultivated Acanthamoeba and Hartmannella for commonly applied isoenzymatic typing procedures. It also describes differences between several species of axenically and monoxenically cultivated Acanthamoeba and Hartmannella.

10 Chapter 7 describes differences between the bacteriolytic activities of species of both genera towards cells of various bacteria. Specific bacteriolytic activities were determined for each amoebal-bacterial combination using a spectrophotometrical assay. Also isoelectric focusing gel-electrophoresis in combination with a newly developed agar-bacteria overlay technique were used to study differences between bacteriolytic patterns. Chapter 8 describes the small-subunit ribosomal RNA (srRNA) analysis of three Hartmannella vermiformis strains from different geographic regions. The primary sequence data were used to obtain the secondary structure of the gene, to determine the differences for this gene between Acanthamoeba and Hartmannella, and to perform phylogenetic analyses. The srRNA data revealed the phylogenetic position of both genera of lobose amoebae among the ciliates, flagellates and other eukaryotes.

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14 61 Moura H., Wallace S. & Visvesvara CS. 1992 Acanthamoeba healyi N Sp and the isoenzyme and immunoblot profiles of Acanthamoeba spp, group I and 3 J. Protozoo! 39 573-583 62 Muller, M. 1967 Digestion ρ 351-380 In Florkin, M & Scheer, BT (eds), Chemical Zoology Vol I Protozoa Academic Press, New York 63 Nahapetian, K., Challemel, O, Beurtin, D., Dubrou, S. Gounon, P. & Squinazi, F. 1991 The intracellular multiplication of Legionella pneumophila in protozoa from hospital plumbing systems Res Microbiol 142 677-685 64 NeiT, RJ. 1957 Purification, axenic cultivation and description of a soil amoeba, Acanthamoeba sp J Protozool 4 176-182 65 Niederkorn, J.Y., Ubelaker, J.E., McCulley, J.P., Stewart, G.L., Myers, D.R.,

Mellon, J.A., Silvany, R.E., He, Y4 Pidherney, M., Martin, J.H. & Alizadeh, H. 1992 Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellami Invest Ophtalmol Vis Set 33 104-112 66 Nisbet, B. 1984 Nutrition and feeding strategies in protozoa Croom Helm, London 67 Page, F.C. 1983 Marine gymnamoebae Cambridge Institution of Terrestrial Ecology 68 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae In Culture Collection of Algae and Protozoa, Freshwater Biological Association The Ferry House, Ambleside, Cumbria 69 Page, F.C. & Siemensma, F J. 1991 Nackte Rhizopoda und Heliozoea Gustav Fisher Verlag, Stuttart 70 Paszko-Kolva, C, Yamamoto, H., Morris, G., Shahamata, M., Colwell, R., Brier, J.W., Tran, T.T. & Sawyer, Т.К. 1990 Microbial contaminants of eyewash stations ATCC Quaterly Newsletter 10 5-7 71 Pernin P., Cariou M-L. & Jaquiera A. 1985 Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naeglena J Protozool 32 592-603 72 Pollard, T.D. 1976 Cytoplasmic contractile proteins J Cell Biol 91 1565-1655 73 Prescott, D.M. & T.W. James. 1955 Culturing of Amoeba proteus on Tetrahymena Exptl Cell Res 8 256-258 74 Rowbotham, Τ J. 1980 Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae J Clin Pathol 33 1179-1183 75 Sanden, G.N., Morrill, W.E., Fields, B.S., Breiman, R.F. & Barbaree, J.M. 1992 Incubation of water samples containing amoebae improves detection of Legtonellae by the culture method Appi Environ Microbiol 58 2001-2004 76 Schaefler, A.A. 1926 Taxonomy of the amoebas with description of thirty-nine new marine and freshwater species Washington Papers from the department of manne biology of the Carnegie Institution 24 77 Schlegel, M. 1991 Protist evolution and phylogeny as discerned from small-subunit nbosomal RNA sequence comparison Europ J Protistol 27 207-219

15 78 Schuster, F.L. 1979 Small amebas and ameba flagellates ρ 21S-28S In Levendowski, M & Hunter, S H (eds ), Biochemistry and physiology of protozoa Vol I Academic Press New York 79 Schuster, F.L. 1990 Phylum Rhizopoda ρ 3-18 In Margulis, L, Corhs, JO, Melkoman, M & Chapman, DJ (eds), Handbook of protoctista The structure, cultivation, habitats and life histones of the eukaryotic microorganisms and their descendants exclusive of animals, plants and fungi Jones and Bartlett Publishers, Boston 80 Shukla, O.P., Kaul, S.M. & Mehlotra, R.K. 1989 Development of improved media for axenic cultivation of Acanthamoeba culbertsom Singh and Das 1970 Indian J Exptl Biol 27 785-791 81 Shukla, O.P., Kaul, S.M. & Mehlotra, R.K. 1990 Nutritional studies on Acanthamoeba culbertsom and development of chemically defined medium J Protozool 37 237-242 82 Siemensma, F.J. 1987 De nederlandse naaktamoeben (Rhizopoda, Gymnamoeba) Wetenschappelijke mededelingen К NN V no 181 83 Singh, B.N. 1941 Selectivity in bacterial food by soil amoebae in pure and mixed culture in sterilized soil Ann Appi Biol 28 52-65 84 Singh, B.N. 1942 Selection of bacterial food by soil flagellates and amoebae Ann Appi Biol 29 18-22 85 Sleigh, M.A. 1989 Protozoa and other protists Edward Arnold, London 86 Sogin, M.L. 1989 Evolution of eukaryotic microorganisms and their small-subunit nbosomal RNAs Amer Zool 29 487-499 87 Sogin, M.L. 1991 The phylogenetic significance of sequence diversity and length variation in eukaryotic small-subunit nbosomal RNA coding regions ρ 175-188 In Warren, L & Koprowski, Η (eds ), New perspectives on evolution Wiley-Liss, Ine, New York 88 Stout, J.D. & Heal, O.W. 1967 Protozoa, ρ 149-195 In Burges, A & Raw, R (eds), Soil biology, Academic Press, Ine New York 89 Taylor, W.T. & Sanders, R.W. 1991 Protozoa ρ 37-93 In Thorp, J Η & Covich, A Ρ (eds ), Ecology and classification of North American freshwater invertebrates Academic Press, San Diego 90 Tyndal, R.L. & Domingue, E.L. 1982 Cocultivation of Legionella pneumophila and free-living amoebae Appi Environ Microbiol 44 954-959 91 Tyndal R.Z., Willaert E., Stevens A. & Nicholson A. 1979 Pathogenic and enzymatic characteristics οι Acanthamoeba isolates from cultured tumor cells Protistologtca 15 17- 22 92 Visvesvara, G.S. 1991 Classification of Acanthamoeba Rev Infect Dis 13 S396-372 93 Visvesvara, G.S. & Stehr-Green, J.K. 1990 Epidemiology of free-living ameba infections J Protozool 37 25S-33S

16 94 Vandenesch, F., Surgot, M., Bornstein, N„ Paucod, J.C., M ar met, D., Isoard, P. & Fleurette, J. 1990 Relationship between free amoeba and Legionella studies in vitro and in vivo Zbl Bakt 272 265-275 95 Wadowsky, R.M., Wilson, T.M„ Kapp, NJ., West, AJ., Kuchta, J.M., States, S.J., Dowling, J.N. & Yee, R.B. 1991 Multiplication of Legionella spp in tap water containing Hartmannella vermiformis Appi Environ Microbiol 57 1950-1955 96 Ward, A.P. 1985 Electrophoretic assessment of taxonomie relationships within the genus Acanthamoeba (Protozoa, Amoebida) Arch Protistenk 130 329-342 97 Weekers, P.H.H. & van der Drift, C. 1993 Nitrogen metabolizing enzyme activities in the free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis J Euk Microbiol 40 251-254

17

Chapter 2

Effects of Grazing by the Free-Living Soil Amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis on Various Bacteria

Peter H.H. Weekers, Paul L.E. Bodelier, John P.H. Wijen & Godfried D. Vogels

Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands

(Submitted for publication)

ABSTRACT

Cultures of 10 different bacteria were used to serve as food source for axenically grown Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis. The non-pigmented Enterobacteriaceae Escherichia coli K12 and Klebsiella aerogenes appeared to be excellent food source to all three amoebae. After 15 days the monoxenic incubations containing E. coli K12 as food source yielded approximately 1.0 χ IO6, 1.6 χ IO5 and 1.7 χ IO7 cells per ml for A. castellana, A. polyphaga and H. vermiformis, respectively. K. aerogenes as food source yielded approximately 3.8 χ IO5, 2.1 χ 10' and 2.6 χ ΙΟ6 cells per ml for A. castellana, A. polyphaga and H. vermiformis, respectively. Hardly any growth or ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens which share the presence of pigmented compounds. A low to moderate amoebal growth was observed with Agrobacterium tumefaciens, Arthrobacter simplex, Bacillus megaterium, Bacillus subtilis, Micrococcus luteus and Pseudomonas fluorescens as food

19 source. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general growth of amoebae and ammonium production increased in the order A. polyphaga, A. castellanti and H. vermiformis.

INTRODUCTION.

Protozoa are the most important predators of bacteria in soil [2, 6, 16, 36] and aquatic systems [3, 23, 24]. Because of their predatory activity they play a major role in controlling the bacterial population in soil [1, 4, 20]. The drastic decrease of bacterial numbers in a short time requires a high grazing pressure and large numbers of active predators with high growth rates. Protozoa meet these requirements since they occur in great numbers in soil [14, 15], have rapid growth rates [11, 12], and are able to decrease bacterial numbers drastically [26-28]. For an arable field soil it was calculated that protozoa consumed 150-900 g bacteria χ m"2 χ year'1 [36]. However, not all bacteria seem to be an equally suitable food source for protozoa. Gram-negative bacteria were able to survive in the presence of many protozoa [1] while biologically formed toxins in bacteria may prevent the attack by protozoa [27]. Protozoan grazing stimulates microbial activity and enhances the turnover of nutrients, particulary nitrogen, which would otherwise become immobilized in bacterial biomass [2, 17, 25, 38]. When protozoa feed on bacteria in water, one-third of the nitrogen in the bacterial biomass is converted into protozoan biomass, one-third is excreted as indigestible parts and one-third is excreted as ammonium [19, 22]. The same relations are most likely valid for bacteria and protozoa in soil since these organisms are only active in the soil water phase and therefore play an active role in the control of soil fertility and soil nutrient cycling [21, 34, 35]. Knowledge about the role and importance of protozoa in soil food chains was already available, but was mainly focused on ciliates and flagellates. Microcosm experiments studying the amoebal grazing activity and nitrogen mineralization suggested that amoebae were important in recycling nitrogen accumulated by rhizosphere microorganisms [7, 17, 18]. Food preferences of free-living soil amoebae and nitrogen mineralization as a result of amoebal

20 grazing are still barely examined. Free-living amoebae are the dominant bacterial consumers in soil [5, 16], and are responsible for 60% of the total decrease of the bacterial population [5]. Decreasing bacterial numbers with increasing amoebal numbers have been reported in both field soils [9] and in sterilized soils [10]. Differences in the ability for amoebae to grow on various food bacteria exist [32, 33]. Some bacteria were preferred while others are non- edible even if they were the only food available [31, 32]. In general, amoebae are reported to prefer to feed on Gram-negative bacteria like the Pseudomonads [36] although Gram-positive bacteria were also consumed [31]. However, it was also reported that nor the presence of pigmentation, and certainly not the relation to Gram-staining can account for the selective action by the amoebae [31]. Quantitative results concerning the predator-prey relationship, edibility of various bacteria, the growth capacity and ammonium production of free-living soil amoebae feeding on various bacteria are scarce. The aim of this study was to investigate food preferences of actively grazing Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis in monoxenic cultures, and to quantitate the amoebal growth response and ammonium production in relation to their food source.

MATERIALS AND METHODS

Organisms and cultivation. Acanthamoeba castellami (CCAP strain 1501/1B) and Acanthamoeba polyphaga (CCAP strain 1501/3C) were obtained as axenic cultures from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, UK) and were cultured as reported previously [30]. Hartmannella vermiformis strain Atlanta (strain CDC-19) was obtained as axenic culture from Dr. B.J. Fields (Respiratory Disease Branch, Center for Infectious Disease, Atlanta, USA) and was cultured as reported previously [37]. Exponentially growing amoebal cells were harvested by centrifugation (10 min , 750 χ g), washed (3 times) with and resuspended in 20 mM Neff Amoeba Saline [30]. Agrobacterium tumefaciens, Arthrobacter simplex, Bacillus megatenum, Bacillus subtilis, Chromatium vmosum, Escherichia coli К12, Klebsiella aerogenes, Micrococcus luteus, Pseudomonas fluorescens and Serratia marcescens were cultured overnight in nutrient broth (Oxoid, Basingstoke, UK)

21 at 30CC on a shaking incubator. Bacterial cells were harvested by centrifugation (10-15 min, 25,000 χ g), washed (3 times) with and resuspended in 20 mM buffered Neff Amoeba Saline. Immediately after harvesting the amoebae and bacteria were used to start monoxenic incubations.

Monoxenic incubations. Axenically grown amoebal cells and pure cultures of bacterial cells were inoculated in sterilized 250 ml Erlenmeyer flasks with cotton plugs containing 25 ml Neff Amoeba Saline buffer (pH 6.8, 20 mM) with initial cell densities of 104 amoebae and 109 bacteria per ml. Amoebal and bacterial controls for cell numbers and ammonium concentrations were inoculated in the same way containing either amoebae or bacteria. Monoxenic cultures and controls were incubated on a shaking incubator (25°C, 40 rpm) in the dark for 15 days. Every other day bacterial feed was added (1 ml) at a concentration of 10' cells per ml in order to prevent substrate limitation.

Sampling and analytical procedures. Every other day homogeneous samples of 1 ml were taken under aseptical conditions and immediately used to quantitate amoebal cell numbers using a counting chamber (Biirker-Turk). Ammonium concentrations were determined afterwards using the salicylate method [29].

RESULTS

Acanthamoeba castellana, Acanthamoeba polyphaga and Hartmannella vermiformis were monoxenically incubated with ten bacteria (Table 1). Amoebal and bacterial controls were incubated simultaneously. Every other day samples were taken to determine the amoebal cell numbers and ammonium concentration to reveal amoebal food preferences and ammonium production for amoebae grazing on various bacteria. Examples of amoebal growth responses are shown in Fig. 1 for H. vermiformis grazing on Escherichia coli Kl2 and Agrobacterium tumefaciens. The growth yields and net ammonium production for the predator-prey combinations incubated for 15 days are shown in Table 1 and revealed distinct differences not only between the various bacteria used as feed but also among the amoebae tested. Amoebal growth was

22 detected in all test combinations but E. coli К12 proved to be a far better food source than indigenous soil bacteria like A. tumefaaens, Arthrobacter simplex, Bacillus megaterium, Bacillus subtilis, Klebsiella aerogenes and Pseudomonas fluorescens. Growth yields varied between 0.01 and 167.5 χ IO5 cells per ml for A. polyphaga grazing on С vinosum or S. marcescens, and H. vermiformis grazing on E. colt К12, respectively. In general Hartmannella showed higher growth yields than Acanthamoeba growing on the same bacteria. In accordance with the amoebal grazing activity distinct differences in the amount of ammonium produced for each monoxemc incubation were detected. The ammonium production in the different monoxenic combinations varied between 0.01 and 15.95 mM for A. polyphaga grazing on S. marcescens, and #. vermiformis grazing on E. coll К12, respectively (Table 1).

Table 1. Amoebae cell yield and ammonium production as result of amoebal growth on various food bacteria

Bacteria A castellami A polyphaga Η vermiformis

amoebae ammonium amoebae ammonium amoebae ammonium yield8 (mM) yield" (mM) yield" (mM) (χ 105 per ml) (x 105 per ml) (x 10' per ml)

A tumefaaens 0 55 ±0 30 1 66 ± 0 10 0 45 ±0 12 1 55 ±0 06 10 84 ±0 84 2 65 ± 0 24 A simplex 031 ±019 0 59 ±0 08 0 70 ±0 17 173 ±0 12 12 21 ± 160 3 93 ± 0 26 В megatenum 1 06 ± 0 14 182 ±0 16 0 76 ± 0 22 108 ±0 18 32 05 ± 3 65 6 44 ± 0 54 В subtilis 2 94 ±0 13 144 ±0 21 0 98±0 14 0 51 ±0 07 32 22 ± 2 43 3 35 ±0 61 С vinosum 0 02 ±0 01 0 43 ±0 18 0 01 ±0 01 0 35 ± 0 09 0 62 ± 0 08 0 91 ±0 02 E coli K12 10 46 ±0 44 11 12 ± 041 1 60 ± 0 27 1 73 ± 0 19 167 5 ± 156 15 95 ±091 К aerogenes 3 77 ± 0 45 4 75 ±0 32 2 08 ±0 37 3 87 ± 0 22 25 55 ± 1 18 6 18 ±0 52 M luteus 0 10 ±0 02 2 47 ±0 19 051 ±011 3 54±0 12 0 35 ±0 10 1 00 ± 0 04 Ρ fluorescens 1 44 ± 0 24 1 27 ± 0 22 1 16 ± 0 31 I 52 ±0 16 11 80 ± 1 42 2 91 ±0 26 S marcescens 0 02 ±0 01 0 62 ± 0 07 001 ±001 001 ±001 1 90 ± 0 33 0 60 ± 0 08

Incubations in 25 ml medium for 15 d and data refer to increase in cell numbers means ± SD in which n=3 for individual experiments " initial amoebal density was 0 1 χ 105 cells per ml values are corrected for bacterial controls

23 100 200 300 400 0 100 200 300 400 Incubation time (hours)

Fig. 1. Typical growth yield and ammonium production curves for monoxenically growing Hartmannella vermiformis on (A) Escherichia coli K12 and (B) Agrobactenum tumefaciens as food bacteria. Amoebal cell numbers (D) and ammonium concentrations (•) are represented as means (n=3 for individual experiments) in which the vertical bars represent the SD.

DISCUSSION

Protozoa, and especially amoebae, were recognized as the major predators of bacteria in soil, thereby regulating the size of the bacterial population. Amoebal grazing on various bacteria during a 2-week period in monoxenic incubation revealed food preferences as expressed in differences in the amoebal growth yield and concomitant ammonium production depending on the type of

24 bacterial food source. The non-pigmented Enterobacteriaceae Escherichia coli К12 and Klebsiella aerogenes appeared to be a far better food source to all three amoebae than the other bacteria tested. Hardly any growth and ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens which share the presence of pigmented compounds. Agrobactenum tumefaciens, Arthrobacter simplex, Bacillus megaterium, Bacillus subtilis, Micrococcus luteus and Pseudomonas fluoresceins, of which some are indigenous soil bacteria, supported a low to moderate growth of the three amoebae. In general growth of amoebae and ammonium production increased in the order Acanthamoeba polyphaga, Acanthamoeba castellami and Hartmannella vermiformis. Differences in growth yield of amoebae were due to the ability to select their food among different kinds of bacteria [13, 32, 33] and might be explained either by the incapabilities of the amoebae in the uptake or digestion of specific bacteria, or by the capability of the bacteria to prevent grazing by means of defense mechanisms, like toxic pigments or special outer-membrane structures. Such mechanisms may hamper extensive grazing on bacteria known to be indigenous in soil, like Gram-negative Pseudomonas and Agrobactenum, and Gram-positive Arthrobacter and Bacillus [8, 36]. Predator-prey relationships can have positive as well as negative effects on the prey organisms. Bacterial numbers may decrease in the presence of amoebae by the consumption of edible bacteria, whereas on the other hand, bacterial numbers of inedible bacteria may increase by a higher rate of mineralization of nutrients in the presence of amoebae [2, 7, 17, 25]. Amoebae feeding on bacteria improved the mineralization of nitrogen from soil organic matter, which previously was immobilized in bacteria, by stimulating the turnover of bacterial biomass [38]. Therefore, grazing of rhizosphere microorganisms, especially bacteria, by the free-living soil amoebae has practical importance for agriculture and forestry.

ACKNOWLEDGEMENTS

This research was supported by a grant from the Netherlands Integrated Program for Soil Research.

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1 Alexander, M. 1981 Why microbial predators and parasites do not eliminate their prey and host Ann Rev Microbiol 35 113-133 2 Bamforth, S.S. 1985 The role of protozoa in litters and soils J Protozool 32 404-409 3 Barcina, L, Ayo, B„ Muela, A„ Egea, L. & Iríberrí, J. 1991 Prédation rates of flagellate and cilliated protozoa on bactenoplankton in a nver FEMS Microb Ecol 85 141-150 4 Casida, L.E. 1989 Protozoan response to the addition of bacterial predators and other bacteria to soil Appi Environ Microbiol 55 1857-1859 5 Clarholm, M. 1981 Protozoan grazing of bacteria in soil - impact and importance Microb Ecol 7 343-350 6 Clarholm, M. 1984 Heterotrophic, free-living protozoa neglected microorganisms with an important task in regulating bacterial populations ρ 321-326 In Klug, M J & Reddy, С A (eds ), Current perspectives in microbial ecology Amer Soc Microb, Washington, D С 7 Clarholm, M. 1985 Possible roles of roots, bacteria, protozoa and fungi in supplying nitrogen to plants ρ 355-365 In Fitter, AH, Atkinson, D, Read, DJ & Usher, MB (eds ), Ecological interactions in soil Spec Pubi No 4, Br Ecol Soc, Blackwell Scientific Publications Ltd , Oxford 8 Coleman, D.C., Cole, C.V., Hunt, H.W. & Klein, D.A. 1978 Trophic interactions in soils as they affect energy and nutrient dynamics I Introduction Microb Ecol 4 345- 349 9 Cutler, D.W., Crump, L.M. & Sandon, H. 1922 A quantitative investigation of the bacterial and protozoan population of soil, with an account of the protozoan fauna Philos Trans R Soc Lond [Biol ] 211 217-250 10 Cutler, D.W. 1923 The action of protozoa on bacteria when inoculated in sterile soil Ann Appi Biol 10 137-141 11 Cutler, D.W. & Crump, L.M. 1927 Soil protozoa and bacteria in relation to their environment J Quekelt Microsc Club Ser 2 15 309-330 12 Cutler, D.W. & Crump, L.M. 1927 The qualitative and quantitative effects of food on the growth of a soil amoeba {Hartmannella hyalma) Br J Exp Biol 5 155-165 13 Danso, S.K.A. & Alexander, M. 1975 Regulation of prédation by prey density the pTOtozoan-Rhizobwm relationship Appi Microb 29 515-521 14 Darbyshire, J.F. & Greaves, M.P. 1973 Bacteria and protozoa in the rhizosphere Pestic Set 4 349-360 15 Darbyshire, J.F. & Greaves, M.P. 1967 Protozoa and bacteria in the rhizosphere of Sinapis alba L, Trifolium repens L and Lolium perenne L Can J Microbiol 13 1057- 1068

26 16 Elliot, E.T. & Coleman, D.C. 1977 Soil protozoan dynamics in a shortgrass prairie Soil Biol Biochem 9 113-118 17 Elliot, E.T., Coleman, D.C & Cole, C.V. 1979 The influence of amoebae on the uptake of nitrogen by plants in gnotobiotic soil ρ 221-229 In Harley, JL & Russell, R S (eds ), The soil-root interface, Academic Press, London 18 Elliot, E.T., Cole, C.V., Coleman, D.C, Anderson, R.V., Hunt, H.W. & McClellan, J.F. 1979 Amoebal growth in soil microcosms a model system of C, N and Ρ trophic dynamics Intern J Environm Studies 13 169-174 19 Fenchel, T. 1982 Ecology of heterotrophic microflagellates II Bioenergetics and growth Mar Ecol Prog Ser 8 225-231 20 Fenchel, T. 1987 Ecology of protozoa the biology of free-living phagothropic prohsts Science Tech Publishers, Madison, Wise USA 21 Foster, R.C. & Dormaar, J.F. 1991 Bacteria-grazing amoebae in situ in the rhizosphere Biol Feml Soils 11 83-87 22 Goldman, J.C., Caron, D.A, Andersen, D.K. & Dennett, M.R. 1985 Nutrient cycling in microflagellate food chains I Nitrogen dynamics Mar Ecol Prog Ser 24 231-242 23 Gonzales, J.M., Iriberri, J., Egea, L. & Barcina, I. 1990 Differential rates of digestion of bacteria by freshwater and marine phagotropic protozoa Appi Environ Microbiol 56 1851-1857 24 Gonzales, J.M., Iriberri, J. Egea, L. & Barcina, I. 1992 Characterisation of culturability, protistan grazing, and death of enteric bacteria in aquatic ecosystems Appi Environ Microbiol 58 998-1004 25 Griffiths, B.S. 1990 A comparison of microbial-feeding, nematodes and protozoa in the rhizospere of different plants Biol Fértil Soils 9 83-88 26 Habte, M. & Alexander, M. 1977 Further evidence for the regulation of bacterial populations in soil by protozoa Arch Microbiol 113 181-183 27 Habte, M. & Alexander, M. 1978 Mechanisms of persistence of low numbers of bacteria preyed upon by protozoa Soil Biol Biochem 10 1-6 28 Habte, M. & Alexander, M. 1978 Protozoan density and the coexistence of protozoan predators and bacterial prey Ecology 59 140-146 29 Kempers, A J. & Zweers, A. 1986 Ammonium determination in soil extracts by the salicylate method Soil Sci Plant Anal 17 715-723 30 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae ρ 16-17 Culture Collection of Algae and Protozoa, Freshwater Biological Association, Ambleside, Cumbna. 31 Singh, B.N. 1941 Selectivity in bacterial food by soil amoebae in pure and mixed culture in sterilized soil Ann Appi Biol 28 52-65 32 Singh, B.N. 1942 Selection of bacterial food by soil flagellates and amoebae Ann Appi Biol 29 18-22

27 33 Singh, B.N. 1946 A method of estimating the numbers of soil protozoa, especially amoebae based on their differential feeding on bacteria Ann Appi Biol 33 112-119 34 Stout, J.D. 1973 The relationship between protozoan populations and biological activity in soils Amer Zool 13 191-201 35 Stout, J.D. 1980 The role of protozoa in nutrient cycling and energy flow Adv Microb Ecol 4 1-50 36 Stout, J.D. & Heal, O.W. 1967 Protozoa ρ 149-195 In Burges, A & Raw, F (eds ), Soil biology. Academic Press, Ine New York 37 Weekers, P.H.H. & van der Drift, C. 1993 Nitrogen metabolizing enzyme activities in the free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis J Euk Microbiol 40 251-254 38 Woods, L.E., Cole, C.V., Elliott, E.Y„ Anderson, R.V. & Coleman, D.C. 1982 Nitrogen transformation in soil as affected by bactenal-microfaunal interactions Soil Biol Biochem 14 93-98

28

Chapter 3

Mass Cultivation of the Free-Living Soil Amoeba, Acanthamoeba castellami in a Laboratory Fermentor

Peter H.H. Weekers, John P.H. Wijen, Bart P. Lomans & Godfried D. Vogels

Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands

(Submitted for publication)

ABSTRACT

Axenical cultivation of Acanthamoeba castellami in laboratory fermentors (14 1) yielded after 20 days approximately 3 g cells (wet weight). After a short lag phase amoebal cell numbers increased exponentially to a maximum of 3.5 χ IO5 cells per ml until cell death occurred after 20 days. Optical density and protein concentrations revealed identical patterns. During amoebal growth only 12-19% of the initially added glucose (100 mM) as sole carbon source was used. Large amounts of ammonia (1 g in 10.5 1 culture volume) were excreted into the medium which subsequently raised the pH from 6.6 to 7.7, and from 6.6 to 6.8 in 2 and 20 mM buffered media, respectively. Growth inhibition and cell death could not be explained by a depletion of glucose or oxygen limitation during growth. The production of ammonia had a growth inhibitory effect; however, the sudden termination of the exponential growth phase and cell death could not be explained by the toxic influence of ammonia only.

31 INTRODUCTION

Various methods have been employed to cultivate free-living soil amoebae. Traditionally amoebae were grown in tap water containing decaying wheat or hay and a variety of microorganisms which served as a food source. These cultures are easy to maintain but produce only a small number of amoebae. Free-living soil amoebae were also grown in buffered mineral salt media containing only one or several species of microorganisms as a food source which are referred to as monoxenic and polyxenic cultures, respectively [20]. Most frequently bacteria, but also small flagellates or ciliates, were used to feed these amoebae [5, 16, 27]. For many purposes the ideal culture is axenic, containing no other species, in which the amoebae absorb only dissolved nutrients. Axenic Acanthamoeba cultures were established in rich complex media [11, 14, 18, 28] and only a few Acanthamoeba species were reported to grow in chemically defined media [1, 9, 29]. Usually amoebae were grown axenically in small batch cultures (3-5 ml), sometimes in larger cultures (up to approximately 400 ml). The only mass cultures of free-living soil amoebae were reported for monoxenically grown Amoeba proteus using a five-stacked row of baking-dishes each containing 150 ml of culture medium [15], and for Acanthamoeba polyphaga growing on bacteria in the second stage of a simplified predator-prey system in a continuous culture [30]. We report on a practical method for the mass cultivation of axenically grown Acanthamoeba castellana in a laboratory fermentor. During the growth process cell numbers, optical density and protein concentrations were determined to quantitate amoebal growth. Specific activities for the glucose consumption, respiration and ammonia production were determined as parameters for the growth process.

MATERIALS AND METHODS

Amoeba strain and cultivation. Acanthamoeba castellana (strain CCAP- 1501/1B) was obtained as axenic culture from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, Cumbria, UK) and was cultured in proteose peptone glucose (PPG-medium) at pH 6.5 as reported previously [20]. Cells

32 were precultured in 500 ml PPG-medium in Fembach flasks (30°C) until a density of approximately 105 cells per ml. These exponential growing cells (1000 ml) were harvested and used to inoculate 10 1 PPG-medium (30°C, pH 6.5) in a 14 1 laboratory scale fermentor (model GF.0014; Chemap AG, Mannedorf, Switzerland). The buffer concentrations applied in the PPG-media were 2 and 20 mM phosphate.

Fermentor design. The fermentor was equipped with special air inlet devices providing micro-bubble aeration and the air flow was regulated with a gas flow controller (Sho-Rate model 1355; Brooks, Hatfield, USA). Sterile air was going in, and processed air was going out the fermentor through sterile 0.2 μηι pore size ceramic filter elements (Schumalith model SC-10; Schumacher, Crailsheim, Germany). The outlet air was bubbled through wash bottles containing H2S04 to trap gaseous ammonia in the processed air. A stirring device and a baffled fermentor vessel were used to establish a homogeneous cell suspension. An oxygen probe (model IL-530; Ingold, Urdorf, Switzerland) connected with a fermentor control (p.e.c. 02-amplifier model 531; Metrohm, Herisau, Switzerland) and a pH probe (model 465-50-90-TS7; Ingold, Urdorf, Switzerland) connected with a pH control (p.e.c. Regler/Controller E450 and Verstärker E370A; Metrohm, Herisau, Switzerland) attached to recorders were used to measure the oxygen concentration and pH changes on-line during the growth process.

Sampling and analytical methods. Each day samples were taken under aseptical conditions and were used immediately to determine the amoebal cell numbers (Bürker-Turk counting chamber), optical density (at 550 nm) and pH. The respiration rates from fresh samples were determined using a Biological Oxygen Monitor (Biowatch 1032 Biocontroller; Applikon, Schiedam, The Netherlands). Glucose concentrations were determined using a standard enzymatic glucose assay (Sigma procedure #510; Sigma, St. Louis, MO, USA). Ammonia/ammonium concentrations were determined by means of a standard enzymatic ammonium assay (Test combination Urea/Ammonia, cat.nr. 542.946; Boehringer Mannheim, Almere, The Netherlands). The protein contents were determined by means of a standard protein assay

33 with bovine γ-globulin as standard (Protein assay kit I, cat nr.: 500-0001; Bio- Rad, Veenendaal, The Netherlands). For all spectrophotometrical assays a Hitachi spectrophotometer was used (digital spectrophotometer model 191; Hitachi, Tokyo, Japan).

RESULTS

Amoebal cell growth. Acanthamoeba castellami cells were grown in a laboratory scale fermentor until cell growth stopped and total cell numbers declined. After a lag phase of approximately 5 days amoebal cell numbers increased exponentially for 15 day until they reached a maximum of approximately 3.5 χ 105 cells per ml (Fig. 1A).

Table 1. Variation in generation times for axenic Acanthamoeba castellami during growth in a laboratory fermentor.

Growth phase Buffer (mM) Generation time (h)

laga 2 115 ±44 20 153 ± 5 exponential15 2 90 ± 7 20 67 ± 14 death0 2 -121 ± 12 20 - 96 ± 10

Mean value ± standard deviation with an=6,n= 10;cn = 5

Generation times varied between 67 and 153 h for different stages of the growth process (Table 1). Approximately three weeks after inoculation the number of viable amoebal cells rapidly declined, however cyst formation was not observed. A similar increase and rapid decline was observed for the OD (at 550 nm) and protein concentration (Fig. IB and 1С).

34 0 30 40x10' В О) 0 25 "È с о ó ** 020 « 015 g •Í л α a A î ^ о io S / # a. / > О / >. 0 05 •_^~" >- • 0 250

200 1 fi σι a. 150 /• '>

100

05х10э - ríi 50

О 10 15 20 25 30 0 5 10 15 20 25 30 incubation time (days)

Fig. 1. Growth of axenic Acanthamoeba castellami represented as; (A) amoeba cell numbers, (B) OD550 and (C) protein concentration, (a) and (•) represent cultures with 2 mM and 20 mM phosphate buffer. Vertical bars indicate the SD of the mean.

Glucose consumption. For cultivation of A. castellami in a proteose peptone medium high concentrations of glucose (80-100 mM) as sole carbon source were required to support optimal growth. As a result of amoebal growth the glucose concentration of the medium decreased from the initial 100 mM to approximately 81 and 88 mM at the end of the cultivation process in the 2 and 20 mM buffered media, respectively (Fig. 2). Specific glucose consumption rates for each growth phase revealed minute differences (Table 2); however, the values for the lag phase were larger.

35 100

95

E. § 90 ra с ω о с о Ü ω oc m 85 о о JD dì

80

75 0 5 10 15 20 25 30 incubation time (days)

Fig. 2. Glucose consumption of axenic Acanthamoeba castellami during the growth process. Glucose concentrations for 2 mM (D) and 20 mM (•) buffered media.

Respiration. The obligate aerobic A. castellami required oxygen for vegetative multiplication. During the growth process the oxygen tension in the medium was kept above 90 percent oxygen saturation by increasing the air supply daily, thus preventing oxygen limitation and subsequent cyst formation. No cysts were detected during the cultivation process. Freshly taken samples showed specific respiration rates which varied between 1.17 and 4.56 mmol oxygen χ mg protein"1 χ h"1 for the different growth phases (Table 2). Although

36 fairly constant values were found for cells in the exponential and death phase, values for lag phase were about 3 times higher.

Table 2. Specific activities for glucose consumption, respiration and ammonium production for axenic Acanthamoeba castellami grown in a laboratory scale fermentor.

Growth Buffer Specific glucose Respiration Specific ammonium phase (mM) consumption rate production + (mmol glucose χ mg (mmol oxygen χ mg (mmol NH4 χ mg protein" χ h ) protein" χ h ) protein" χ h ) lag" 2 2 436 ±0 057 4 55 ± 1 26 0 233 ±0 055 20 0 827 ±0 215 4 56 ± 0 76 0 200 ± 0 034 exponential 2 0486±0310 1 63 ± 0 22 0 158 ±0 024 20 0 269 ±0 100 1 35 ±0 12 0 072 ±0 014 deathc 2 0 439 ±0 176 1 49 ± 0 33 0 015 ±0 005 20 0 398 ± 0 079 1 17 ± 0 12 0 041 ±0 020

Mean value ± standard deviation with a η = 6, b η 10, c η = 5

Ammonia production and pH. Increasing amounts of ammonia (up to 5 mM) accumulated in the medium as a result of cell growth (Fig. 3). A. castellami grown in 10.5 1 culture volume produced 1.00 and 0.93 g ammonia of which 3.5% and 0.5% was trapped in wash bottles in the 2 and 20 mM buffered cultures, respectively. Specific ammonia production rates were determined and varied between 0.015 and 0.233 mmol NH/ χ mg protein"1 χ h'1 (Table 2). No changes in pH were observed during the lag phase and early- exponential phase for both 2 and 20 mM phosphate buffered media. However, due to the ammonia production the pH in the mid-exponential growth phase of the 2 mM buffered media increased rapidly from 6 6 to 7.7, while in media with 20 mM buffer concentration the increase in pH (6.6 to 6.8) was much smaller (Fig. 3).

37 85

β.3

ryOQ-D-Ο-Π < ^ 8.1

79

7.7

/ 7.5

/ / 7 7.3 7.1 'V > imK¿&- 69 A¿* 6.7

6.5 10 15 20 25 30

incubation time (days)

Fig. 3. Ammonium concentration (D , •) and pH (Δ , A) during the growth process of axenic Acanthamoeba castellana. Open and closed symbols represent 2 and 20 mM buffered media, respectively.

DISCUSSION

Cell growth and generation time. Acanthamoeba castellami was the first free-living soil amoeba successfully cultivated in an axenic medium [18]. Generation times of 6-20 hours were achieved in small batch cultures (5 ml) when Acanthamoeba was grown in monolayers [8]. However, by using these culturing methods only small quantities of cells were obtained. We were able to grow large quantities οι A. castellami in a laboratory scale fermentor (14 1) to yield after a growth period of 20 days approximately 3 g cells (wet weight). After a lag phase of a few days the generation time for A. castellami in the

38 exponential phase was 67 and 90 hours depending on the buffer concentration. These values fit well in the range of generation times previously reported for various Acanthamoeba species, which varied between 40 and 200 hours for small scale cultures (up to 400 ml) and of which the variation in generation times were explained by differences in growth medium, growth conditions and culturing volumes [1-4, 7, 9, 13, 16, 24, 28, 29].

Growth parameters. During growth of A. castellami in a fermentor glucose consumption, respiration and ammonia production were used as parameters to describe the growth process. Most Acanthamoeba sp. were only able to grow in proteose peptone (PPG- medium) if relatively high glucose concentrations (80-100 mM) were used. Despite the high glucose concentrations in PPG-media [10, 19] and also in other glucose containing growth media [1, 3, 4, 12, 13, 29], glucose consumption has only been described for Hartmannella rhysodes (now classified as Acanthamoeba rhysodes) [7]. We observed a decrease in the initial 100 mM glucose concentration of 12-19% during growth in the fermentor. These values fit in the range previously reported for small batch cultures (5 ml) of H. rhysodes grown in PPG-media containing 100 mM glucose [7]. Specific glucose consumption rates (Table 2) were fairly constant indicating that there is no limitation of glucose during cell growth, even not in the death phase. Respiration rates were determined for cell samples taken from the fermentor and amounted to 1.63 and 1.35 mmol oxygen χ mg protein"1 χ h"1 for exponentially growing cells in 2 and 20 mM buffered media, respectively. Respiration rates determined for batch cultures (400 ml) of A. castellana showed respiration rates of approximately 1.25 mmol oxygen χ mg protein"1 χ h"1 [unpublished results]. Both rates fit in the range (0.43-1.56 mmol oxygen χ mg protein'1 χ h') previously reported for exponentially growing Acanthamoeba [6, 8, 14]. Ammonia is known to be an important product of free-living protozoa. Specific ammonia production rates were determined for A. castellami (Table 2) and were compared with data from Acanthamoeba sp. grown in batch cultures. The specific ammonia production rates in the exponential growth phase for A. castellami grown in 2 and 20 mM buffer were 0.158 and 0.072 mmol ammonia χ mg protein"1 χ h"1, respectively. For small batch cultures (400 ml) of A.

39 castellami and A. polyphaga 0.19 and 0.13 mmol ammonia χ mg protein"1 χ h"1 were produced, respectively [unpublished results].

Growth inhibition and cell death. At the end of the exponential phase amoebal growth was inhibited and cell numbers declined rapidly although sufficient glucose was still available. Oxygen deficiencies, reported to be a likely cause for growth termination [8, 14], played no role since the oxygen tensions in the fermentor were kept above 90% oxygen saturation. Ammonia was reported as the major source of toxicity in Tetrahymena cultures [17]. In small batch cultures we observed an inhibition of amoebal growth of 59% and 65% if ammonia in an initial concentration of 5 and 10 mM, respectively, was added to the growth media prior to inoculation [unpublished results]. Perhaps the sudden termination of the exponential phase is affected by an unknown factor. The hypothesis of Pigon [21-23, 25, 26] that termination of exponential growth and cell death were induced by a thermolabile growth-inhibiting factor could explain this phenomenon. However, insufficient data are available to indicate which factor could be primarily responsible for the termination of exponential growth.

ACKNOWLEDGEMENTS

We thank W.J. Geerts for technical assistance in operating the large scale fermentors. This research was supported by a grant from the Netherlands Integrated Program for Soil Research.

LITERATURE CITED

1 Adam, K.M.G. 1959 The growth of Acanthamoeba sp in a chemically defined medium J Gen Microbiol 21 519-529 2 Adam, K.M.G. 1964 A comparative study of Hartmannehd amoebae J Protozoo] 11 423-430 3 Adam, K.M.C. 1964 The amino acid requirements of Acanthamoeba sp Neff J Protozoo! 11 98-100

40 4 Adam, K.M.G. & Blewett, DA. 1967 Carbohydrate utilization by the soil amoeba Hartmannella castellami J Protozool 14 277-282 5 Andresen, N. 19S6 Cytologic^! investigations on the giant amoeba Chaos chaos Compi Rend Lab Carlsberg Ser Chim 29 435-555 6 Baldock, B.M., RogersoD, A. & Berger, J. 1982 Further studies on respiratory rates of freshwater amoebae (Rhizopoda, Gymnamoebia) Microb Ecol 8 55-60 7 Band, R.N. 1959 Nutritional and related biological studies on the free-living soil amoeba, Hartmannella rhysodes J Gen Microbiol 21 80-95 8 Byers, TJ. 1979 Growth, reproduction and differentiation in Acanthamoeba Int Rev Cytol 61 283-338

9 Byers, TJ4 Akin, RA., Maynard, В J., Lefken, RA. & Martín, S.M. 1980 Rapid growth of Acanthamoeba in defined media, induction of encystment by glucose-acetate starvation J Protozool 27 216-219 10 Cailleau, R. 1933 Culture a'Acanthamoeba castellami sur milieu peptone Action sur les glucides Compi Rend Soc Biol Pans 114 474-476 11 Chagla, A.H. & Griffiths, A.J. 1978 Synchronous cultures of Acanthamoeba castellami and their use in the study of encystation J Gen Microbiol 108 39-43 12 Chapman-Andresen, С. & Holier, H. 1964 Differential uptake of protein and glucose by pinocytosis in Amoeba proteus Compi Rend Trav Lab Carlsberg 34 211 -226 13 Dolphin, W.D. 1976 Effect of glucose on glycine requirement of Acanthamoeba castellanti J Protozool 23 455-457 14 Edwards, S.W. & Lloyd, D. 1977 Changes in oxygen uptake rates, enzyme activities, cytochrome amounts and adenine nucleotide pool levels during growth of Acanthamoeba castellami in batch cultures J Gen Microbiol 102 135-144 15 Goldstein, L. & Ко, С. 1976 A method for the mass cultunng of large free-living amoebas ρ 239-246 In Prescott, D M (ed ), Methods in cell biology Academic Press, New York 16 Griffin, J.L. 1960 An improved mass culture method for the large free-living amoebae Exptl Cell Res 21 170-178 17 Larsen, J., Svensmark, В. & Nilsson, J.R. 1988 Variation in the growth medium

during the culture cycle of Telrahymena with special reference to ammonia (ΝΗ3), ammonium (NH/), and pH J Protozool 35 541-546 18 Neff, R.J. 1957 Purification, axenic cultivation and description of a soil amoeba, Acanthamoeba sp J Protozool 4 176-182 19 NefT, RJ., NefT, R.H. & Taylor, R.E. 1958 The nutrition and metabolism of a soil amoeba, Acanthamoeba sp Physiol Zool 31 73-91 20 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae In Culture Collection of Algae and Protozoa, Freshwater Biological Association The Ferry House, Ambleside, Cumbria

41 21 Pigon, Α. 1970 Hanmannella culture growth and cell substratum contact Exptl Ceti Res 63 443-447 22 Pigon, A. 1970 Hartmannella growth controlling substances in culture medium Protoplasma 70 405-414 23 Pigon, A. 1972 Inhibition of movement, attachment, and cytokinesis by autogenous substances in the amoeba Hartmannella Exptl Cell Res 73 170-176 24 Pigon, A. 1976 Cell surface and medium conditioning in Acanthamoeba cultures Cytobiologie 13 107-117 25 Pigon, A. 1978 Origin of medium exudate and its effect on cell surface in Acanthamoeba cultures. Cytobiologie 16 259-267 26 Pigon, A. 1981 Growth-affecting activity of cellular microexudate of Acanthamoeba Cytobiostt 15-28 27 Prescott, D.M. & James, T.W. 1955 Cultunng of Amoeba proteus on Tetrahymena Exptl Cell Res 8 256-258 28 ShukJa, O.P., Kaul, S.M & Mehlotra, R.K. 1989 Development of improved media for axenic cultivation of Acanthamoeba culbertsont, Singh and Das 1970 Indian J Exptl Biol 27 785-791 29 Shukla, O.P., Kaul, S.M. & Mehlotra, R.K. 1990 Nutritional studies on Acanthamoeba culbertsont and development of chemically defined medium J Protozool 37 237-242 30 Sinclair, M J., McClellan, J.F., Sinclair, J.L. & Coleman, D.C. 1981 Yield constant for Acanthamoeba polyphaga in batch and continuous culture J Protozool 28 479-483

42

Chapter 4

Axenic Cultivation of the Free-Living Soil Amoebae, Acanthamoeba castellami and Hartmannella vermiformis in a Chemostat

Peter H.H. Weekers & Godfried D. Vogels

Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands

(Submitted for publication)

SUMMARY

A simple procedure for the axenic cultivation of the free-living soil amoebae Acanthamoeba castellami and Hartmannella vermiformis in chemostats was developed. Amoebal cell numbers, optical density and protein concentrations were determined to quantitate amoebal cell growth, and respiration rates were determined to assure optimum growth conditions for the cultures. Dilution rates were gradually increased to obtain steady-state conditions with mininum generation times and maximum cell numbers of approximately 25 and 16 hours, and 2.5 and 3.2 χ IO6 cells per ml for A. castellami and H. vermiformis, respectively.

INTRODUCTION

Various methods for the cultivation of free-living soil amoebae have been employed in the past. Traditionally small monoxenic and xenic batch cultures

45 were established in tap water or in buffered mineral salt media containing a variety of microorganisms which served as a food source [10]. Most frequently bacteria, but also small flagellates or ciliates, were used to feed the amoebae [2, 8, 11]. Only Acanthamoeba polyphaga has been reported to grow monoxenically in a chemostat on bacteria in the second stage of a simplified predator-prey system [14]. For many purposes the ideal cultures are axenic in which amoebae only absorb dissolved nutrients. Small batch cultures of axenic Acanthamoeba were established in rich complex media [6, 7, 9, 12] and in chemically defined media [1, 5, 13]. No continuous cultures for axenically growing amoebae in chemostats have been described. Such cultures may provide high densities of axenic cells in the same growth phase to be used in studies of the physiology and cellular processes of the free-living soil amoebae. This paper describes a simple and practical method for the axenic cultivation of Acanthamoeba castellami and Hartmannella vermiformis in chemostats.

MATERIALS AND METHODS

Amoebal strains and media. Acanthamoeba castellami (strain CCAP- 1501/1B) was obtained as axenic culture from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, Cumbria, UK) and was cultured in proteose peptone glucose (PPG-medium) as reported previously [10]. Hartmannella vermiformis (strain CDC-19) was obtained from Dr. B.J. Fields (Respiratory Disease Branch, Center for Infectious Disease, Atlanta, GA, USA) and was cultured in peptone, yeast extract, nucleic acids, folic acid, hemine (PYNFH- medium) as reported previously [15]. A. castellami was precultured in 50 ml PPG-medium and H. vermiformis in 50 ml PYNFH-medium, both at 30°C and pH 6.5, until a cell density of approximately 105 cells per ml was achieved. Exponentially growing A. castellami and H. vermiformis cells were harvested and used to inoculate a chemostat with 400 ml PPG-medium and 250 ml PYNFH-medium, respectively.

Chemostat design. The chemostat (model: MultiGen; New Brunswick Scientific Co Ine, Edison, NJ, USA) consisted of a baffeled 1 1 culture vessel

46 with two six-bladed flat disc turbine impellers to establish a homogeneous cell suspension. Special air inlet devices provided micro-bubble aeration and a regulated flow of air through sterile filters (Millex-FG^,; Millipore S.A., Molsheim, France). A peristaltic pump (Watson-Marlow model 502S; A. De Jong Т.Н. B.V., Rotterdam, The Netherlands) equipped with sterilized tubing was used to supply the chemostat with influent (fresh medium) and to remove the effluent which was collected in sterilized 1 1 serum flasks.

Sampling and analytical methods. Each day samples were taken under aseptical conditions and were used immediately to determine the amoebal cell numbers (Burker-Turk counting chamber), optical density (at 550 nm) and pH. Freshly taken samples were used to determine respiration rates by means of a Biological Oxygen Monitor (Biowatch 1032 Biocontroller; Applikon, Schiedam, The Netherlands). Protein contents were determined by means of a standard protein assay with bovine γ-globulin as standard (Protein assay kit I, cat nr.: 500-0001; Bio-Rad, Veenendaal, The Netherlands). For all spectrophotometrical assays a Hitachi spectrophotometer was used (digital spectrophotometer model 191; Hitachi, Tokyo, Japan).

RESULTS

Amoebal cell growth. Exponentially growing cells of Acanthamoeba castellami and Hartmannella vermiformis inoculated in chemostats operating at low dilution rates (D = 0.004 and 0.014 h"1, respectively) showed a short lag phase period of a few days (Figs. 1A and 2A). After the adaptation period the amoebal cell numbers started to increase and generation times decreased. Dilution rates were step by step increased until a balanced cell growth and a stable pH was established (Figs. 1A and 2A). Maximum amoebal cell numbers of approximately 2.5 and 3.2 χ IO6 cells per ml (Figs. 1A and 2A) and minimum generation times of 25.2 ± 3.0 and 16.1 ± 0.3 h (Table 1) were reached for A. castellami and H. vermiformis, respectively. Similar results were obtained for changes in the optical density (550 nm) (Figs. IB and 2B) and protein concentrations (Figs. 1С and 2C).

47 Table 1. Dilution rates, generation times and respiration rates for Acanthamoeba castellami during growth in a chemostat culture.

Acanthamoeba castellami IIe IIIe IVa Ve VI1 VII*

Dilution 0.004 0.008 0.012 0.016 0.020 0.024 0.028 rate (h"1) Generation > 250 51.1 46.9 29.0 33.0 27.8 25.2 time (h) ±18.9 ±10.2 ±5.9 ±2.3 ±1.6 ±3.0 Respiration

rate (mmol 02 χ 0.54 2.82 2.59 1.84 1.81 1.79 1.63 mg protein" χ h ) ±0.17 ±0.58 ±0.26 ±0.22 ±0.27 ±0.21 ±0.17

Values for the generation times and respiration rates represent mean ± SD in which a n=7, b n=5, c n=10, ° n=5, e n=9, f n=12 and « n=12

_]__] n| in |ιν* ν ; vi | vu 1 _ _-; 1 0 л**!· 0 f -н^ о in 06 ¿? 6 (Л 3 0 «10 с 01 Ό 04 g

02 *

о - 72 \ ι I и I in ivf ν f vi I Vil С 900 ~ ¿У*Ч** eoo Ê 700 3 - 600 | 500 I 400 8

6 эоо 8 05x10 Ç 200 S 2 л** 100 °- Ч.: -J ' 0 20 30 40 50 0 10 20 30 40 50 60 Time (days) Time (days)

Fig. 1. Growth of axenic Acanthamoeba castellami in PPG-media. (A) amoebae cell numbers (•) and pH (A), (B) ODSSO and (C) protein concentrations. Vertical bars indicate the SD of the mean. Dilution rates are: (I) 0.004, (II) 0.008, (III) 0.012, (IV) 0 016, (V) 0.020, (VI) 0.024 and (VII) 0.028.

48 Table 2. Dilution rates, generation times and respiration rates for Hartmannella vermiformis during growth in a chemostat culture.

Hartmannella vermiformis Hb IIIe IVa Ve

Dilution 0.014 0.022 0.029 0.036 0.044 rate (h"1) Generation 37.4 30.6 22.0 19.4 16.1 time (h) ±8.6 ±6.9 ±3.3 ± 1.5 ±0.3 Respiration

rate (mmol 02 X 2.27 1.63 1.38 1.40 1.33 mg protein' χ h"1) ±0.55 ±0.45 ±0.37 ± 0.12 ±0.15

Values for the generation times and respiration rates represent mean ± SD in which 8 n=7, b n=18, c n=15, d n=8 and e n=13

3 0« 10s

» 2.0 ж 106

* 1.5 м IO6

1.0 x10е -

10 20 30 40 50 60 0 10 20 30 40 50 60 Time (days) Time (days)

Fig. 2. Growth of axenic Hartmannella vermiformis in PYNFH-media. (A) amoebae cell numbers (•) and pH (•), (B) OD550 and (C) protein concentrations. Vertical bars indicate the SD of the mean. Dilution rates are: (I) 0.014, (II) 0.022, (III) 0.029, (IV) 0.036 and (V) 0.044.

49 Respiration rates. Both Acanthamoeba castellana and Hartmannella vermiformis are obligate aerobic amoebae requiring oxygen for vegetative multiplication. During growth the oxygen tension in the medium was kept above 90 percent oxygen saturation by raising the air supply, thus preventing oxygen limitation and subsequent cyst formation. No cysts were detected during the growth in chemostat cultures. Freshly taken samples of A. castellami and H. vermiformis showed fairly constant values for the specific respiration 1 1 rates (appoximately 1.7 and 1.4 mmol 02 χ mg protein' χ h' , respectively) at the highest dilution rates (D = 0.024-0.028 and 0.036-0.044, respectively) which differed only slightly between both amoebae (Table 1 and 2).

DISCUSSION

Acanthamoeba castellami was the first free-living soil amoeba succesfiilly cultivated in an axenic medium [9] and low generation times of 6-20 hours were achieved if cells were grown in monolayer cultures [4]. In this study A. castellami and Hartmannella vermifomxs cells were grown as continuous cultures in chemostats. After an adaptation period of a few days the dilution rate was increased step by step and minimal generation times of approximately 25 and 16 h were achieved for A. castellami and H. vermiformis, respectively. For A. castellami this fits well in the wide range of generation times (6-200 h) previously reported for various Acanthamoeba species growing in suspension cultures [1, 5, 8, 12, 13]. During the balanced growth at the highest dilution rates amoebal cell growth was only limited by the minimum generation time of the cells. Growth was not limited by oxygen depletion or accumulation of toxic ammonia. Respiration rates, used to monitor the cell activity during growth, showed a slight variation for the stages with different dilution rates. For A. castellami these values (Table 1) fall in the same range as respiration rates determined for exponentially growing cells for large batch cultures (400 ml) and laboratory scale fermentor cultures (14 I) which were approximately 1.3 and 1.6 mmol oxygen χ mg protein"' χ h'1, respectively [unpublished results]. These specific respiration rates fall also in the same range (0.43-1.56 mmol oxygen χ mg protein"1 χ h"1) as previously reported [3, 4, 7]. Respiration rates were not

50 available for axenically grown H. vermiformis since this amoeba was only recently axenized The continuous growth has proven to be a successful method for a convenient and simple cultivation of free-living soil amoebae and will facilitate biochemical and physiological investigations.

LITERATURE CITED

1 Adam, K.M.G. 19S9 The growth of Acanthamoeba sp in a chemically defined medium J Gen Microbiol 21:519-529 2 Andresen, N. 1956 Cytological investigations on the giant amoeba Chaos chaos Compi Rend Lab Carlsberg Ser Chim 29:435-555 3 Baldock, B.M., Rogerson, A. & Berger, J. 1982 Further studies on respiratory rates of freshwater amoebae (Rhizopoda, Gymnamoebia) Microb Ecol 8 55-60 4 Byers, Τ J. 1979 Growth, reproduction and differentiation in Acanthamoeba Int Rev Cytol 61 283-338 5 Byers, T.J., Akin, R.A., Maynard, B.J., Lefken, R.A. & Martin, S.M. 1980 Rapid growth of Acanthamoeba in defined media, induction of encystment by glucose-acetate starvation J Protozoo! 27216-219 6 Chagla, A.H. & Griffiths, A.J. 1978 Synchronous cultures of Acanthamoeba castellanti and their use in the study of encystation J Gen Microbiol 108:39-43 7 Edwards, S.W. & Lloyd, D. 1977 Changes in oxygen uptake rates, enzyme activities, cytochrome amounts and adenine nucleotide pool levels during growth oí Acanthamoeba castellami in batch cultures J Gen Microbiol 102 135-144 8 Griffin, J.L. 1960 An improved mass culture method for the large free-living amoebae Exptl Cell Res 21:170-178 9 Neff, RJ. 1957 Purification, axemc cultivation and description of a soil amoeba, Acanthamoeba sp J Protozoo! 4:176-182 10 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae In Culture Collection of Algae and Protozoa, Freshwater Biological Association The Ferry House, Ambleside, Cumbna 11 Prescott, D.M. & James, T.W. 1955 Cultunng of Amoeba proteus on Tetrahymena Exptl Cell Res 8 256-258 12 Shukla, O.P., Kaul, S.M. & Mehlotra, R.K. 1989 Development of improved media for axemc cultivation of Acanthamoeba culbertsont, Singh and Das 1970 Indian J Exptl Biol 27:785-791

51 13 Shukla, O.P., KauI, S.M. & Mehlotra, R.K. 1990 Nutritional studies on Acanthamoeba culbensom and development of chemically defined medium J Protozoo] 37 237-242

14 Sinclair, MJ4 McClellan, J.F., Sinclair, J.L. & Coleman, D.C. 1981 Yield constant for Acanthamoeba polyphaga in batch and continuous culture J Protozoo! 28 479-483 13 Weckers, P.H.H. & van der Drift, С 1993 Nitrogen metabolizing enzyme activities in the free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis J Euk Microbiol 40 251 -254

52

Chapter 5

Nitrogen Metabolizing Enzyme Activities in the Free-Living Soil Amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis

Peter H.H. Weekers & Chris van der Drift

Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands

(Published in the Journal of Eukaryotic Microbiology 1993, 40:251-254.)

ABSTRACT

Free-living soil amoebae consume a wide variety of food including algae, yeast, small protozoa and especially bacteria, which they digest to fulfil their nutritional requirements. Amoebae play an active role in the nitrogen mineralization in soils due to their nitrogen metabolizing capacities. However, little is known about nitrogen metabolizing enzyme activities in these free- living soil amoebae. In this study a number of key enzymes involved in the nitrogen metabolism of the axenically cultivated free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and two different strains of Hartmannella vermiformis were determined. The specific enzyme activities for exponential growth phase cells were calculated and it appeared that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activity. Glutamate synthase activity could not be detected in all species. The levels of specific activities varied depending on the enzymes tested. For all species the highest activities were detected for the

55 transaminase reactions yielding glutamate, and for glutamate dehydrogenase. Differences in specific activities were detected between the species. A general conclusion is that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes.

INTRODUCTION

The free-living soil amoebae are, among the protozoa, assumed to be the organisms having the greatest impact on the mineralization of nitrogen in soils [30]. These protozoan grazers enhance nitrogen mineralization and uptake of nitrogen by plants and the activity of grazers in the rhizosphere, therefore, is directly related to the availibility of nitrogen in soils [14]. Furthermore it has also been suggested that free-living amoebae feeding on bacteria cause a local mineralization of nitrogen close to the plant root [6, 7]. The amoebae are dependent upon amino acids as their primary nitrogen source [1]. In contrast to the parasitic forms, the Hartmannellid soil amoebae do not require any preformed purines or pyrimidines [29]. Until recently, little information has been available concerning nitrogen metabolism in free-living amoebae. Most studies have focused only on the amino acid requirements of these organisms [8, 10, 17, 29]. The reason for this lack of information is that only a few species, mainly Acanthamoeba [4], Naegleria [9], Tetrahymew, trypanosomida and plasmodia [29], can be cultivated in large quantities under axenic conditions. Therefore, data concerning enzymatic capacities obtained from monoxenic or xenic protozoan cultures usually are ambiguous, since the microorganisms used as food obscure the picture. Urate oxidase activities were determined for Polytomella caeca [13], Ochromonas malhamensis [18, 19], Chlamydomonas reinhardtii [2], Tetrahymena pyriformis [15, 25], and the amoebae Chaos chaos, Amoeba proteus [24], Acanthamoeba castellami [23] and Hartmannella culbertsoni [5]. NADP-dependent glutamate dehydrogenase activities have only been reported for Dictyostelium discoideum [27]. The aim of this study was to measure the activity of a number of key enzymes of nitrogen metabolism in axenic cultures of free-living amoebae, in order to obtain insight into the possible role and impact of these microorganisms in the mineralization of nitrogen in soils.

56 MATERIALS AND METHODS

Organisms and cultivations. Acanthamoeba castellami (strain CCAP- 1501/1B) and Acanthamoeba polyphaga (strain CCAP-1501/3C) were obtained as axenic cultures from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, UK). Hartmannella vermiformis strain Atlanta (ATCC strain 50236) was obtained as axenic culture from Dr. B.J. Fields (Respiratory Disease Branch, Center for Infectious Disease; Atlanta, GA, USA) and H. vermiformis strain Koblenz (strain OS-101) was obtained as axenic culture from Dr. R. Michel (Emst-Rodenwaldt Insititut, Koblenz, Germany). All the amoebae cells were cultivated at pH 6.5 and at a temperature of 30°C in a sterilized Peptone Yeast extract, Nucleic acids, Folic acid, Hemin, standard medium (PYNFH- medium), containing 1% bacto peptone (Difco, Brunschwig Chemie, Amsterdam, The Netherlands), 1% yeast extract (Oxoid, Brunschwig Chemie, Amsterdam, The Netherlands), 0.1% ribonucleic acids from yeast (BDH, Basingstoke, UK), 10% foetal calf serum (Gibco BRL, Life Technologies B.V.,

Breda, The Netherlands), 34 μΜ folic acid, 1 μΜ hemin, 2.66 mM KH2P04 and 3.52 mM Na2HP04. Cells were harvested by centrifugation (10 min , 750 χ g) and thoroughly washed using Neff Amoeba Saline [26]. Crude cell homogenates were prepared by freezing/thawing using ethanol/solid carbon dioxide. For preparation of the supernatant the crude cell homogenate was centrifuged in an Eppendorf centrifuge (5 min , 12,000 χ g). To exclude the possibility that NH/ present in the crude extract interfered with the assays, all assays were performed both with crude extract and desalted extract (Econo- Pac P6 cartridge, Biorad, Richmond, CA, USA).

Enzyme assays. Urate oxidase (UO; urate:02 oxidoreductase, EC 1.7.3.3) was assayed spectrophotometrically by measuring the decrease of the absorbance at 283 nm as described by Mahler [20]. For this urate oxidase enzyme assay crude amoebae cell homogenates were used. All other assays were performed using a debris-free supernatant of the crude amoebae cell homogenates in order to measure the activities directly in a cuvette. Glutamine synthetase (GS; L-glutamate:ammonia ligase (ADP), EC 6.3.1.2) was measured spectrophotometrically at 540 nm as described by Ferguson and Sims [11].

57 NADH-dependent glutamate dehydrogenase (NADH:GDH; L-glutamate: NAD oxidoreductase, EC 1.4.1.2), NADPH-dependent glutamate dehydrogenase (NADPH:GDH; L-glutamate:NADP oxidoreductase, EC 1.4.1.4) and glutamate synthase (GOGAT; L-gIutamine:2-oxoglutarate aminotransferase, EC 2.6.1.53) were assayed spectrophotometrically by measuring the NAD(P)H oxidation at 340 nm as described by Meers et al. [21]. Glutamate oxaloacetate transaminase (GOT; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), glutamate pyruvate transaminase (GPT; L- alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2), alanine dehydrogenase (ADH; L-alanine:NAD oxidoreductase, EC 1.4.1.1) and lactate dehydrogenase (LDH; L-lactate:NAD oxidoreductase, EC 1.1.1.27) were assayed spectrophotometrically by measuring the NAD(P)H oxidation at 340 nm as described by Kenealy et al. [16]. The urate oxidase activities and glutamine synthetase were measured on a Hitachi spectrophotometer (digital spectrophotometer model 191; Hitachi, Tokyo, Japan). All other enzyme activities were measured by using a time programmed assay in a kinetics spectrophotometer (Ultraspec К, model 4053; LKB, Bromma, Sweden). The protein content was determined by means of a standard protein assay with bovine γ-globulin as standard (Protein assay kit I, cat nr.: 500-0001; Bio-Rad, Veenendaal, The Netherlands).

RESULTS AND DISCUSSION

The specific activities of the enzymes assayed in the the cell homogenates of Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis (strain Atlanta and Koblenz) are presented in Table 1. Supernatant fractions of the cell homogenates were used except for the urate oxidase. In this assay the whole cell homogenate was used since urate oxidase is a membrane bound peroxisomal enzyme [23, 25]. In all species high specific activities for the enzymes involved in the release of ammonia were detected. Glutamine synthetase and NADH-dependent glutamate dehydrogenase activities in the range of 30-200 χ 10"3 IU χ mg protein"1 and 25-125 χ IO"3 IU χ mg protein'1, respectively, were measured. Due to the activity of these enzymes ammonia can be released and subsequently excreted out of the cell (Fig. 1).

58 Table 1. Specific activities" for enzymes involved in the nitrogen metabolism in free-living soil amoebae.

Enzyme Amoebae strains

A castellami A polyphaga H vermiformis Atlanta Koblenz

UO 25 ± 02 44± 03 14± 02 nd GS 29 4 ± 0 5 207 2 ± 15 312± 03 28 4 ± 0 4 GOT 1255 7 ±41 4 846 9 ± 35 4 325 3 ±16 3 269 5 ± 10 1 GPT 226 3 ±10 7 478 6 ± 18 1 149 9 ± 7 1 103 3 ± 3 1 GOGAT NADH <01 <01 < 0 1 <01 GOGAT NADPH <01 <01 <01 <0 1 GDH NADH 37 6 ± 2 1 24 1 ± 0 6 125 5 ± 4 1 1142 ± 24 GDH NADPH 1 8± 01 38±01 35 0± 0 1 29 4 ± 0 1 ADH NADH <01 <01 <01 <01 ADH NADPH <01 <01 <01 <01

specific activities are expressed as IU χ 10 χ mg protein' and are means ± SD for 5 separate lysates measured in duplicate, nd, not determined, UO, urate oxidase, GS, glutamine synthetase, GDH, glutamate dehydrogenase, GOT, glutamate oxaioacetate transaminase, GPT, glutamate pyruvate transaminase, GOGAT, glutamate synthase, ADH, alanine dehydrogenase

In all species a very low specific activity, less than 10"1 IU χ mg protein*', was detected for both NADH and NADPH-dependent glutamate synthase and both NADH- and NADPH-dependent alanine dehydrogenase. For glutamate synthase this might be expected because the enzyme normally operates under low ammonia concentrations [21]. The amoeba cells are usually grown under high total nitrogen concentrations (150-200 mM total nitrogen) and are not ammonia-limited, but rather produce it as result of their metabolism. A second explanation is that, although freshly made cell extracts were used, glutamate synthase can be inactivated very rapidly even in enzyme-stabilizing buffer solutions [21, 22]. The very low specific activity of alanine dehydrogenase can be explained by the fact that amoebae contain high levels of glutamate

59 pyruvate transaminase activity and therefore do not need alanine dehydrogenase for alanine synthesis.

PROTEIN i proteases _^\. L-aspartate a-ketoglutarate L-alanine

oxaloacetate L-glutamate pyruvate 1 r NAD" NADH NH/

\ EXCRETION

Fig 1. A pathway for ammonia mineralization by amoebae. 1. Glutamate Oxaloacetate Transaminase (GOT), 2. Glutamate Pyruvate Transaminase (GPT) and 3. NADH-dependent Glutamate Dehydrogenase (NADH:GDH).

NADPH-dependent glutamate dehydrogenase has normally anabolic functions while NADH-dependent glutamate dehydrogenase has a catabolic function [28]. Both cofactors, NADH and NADPH, were used to determine whether the

60 glutamate dehydrogenase activity in amoebae was catabolic (NADH-dependent) or anabolic (NADPH-dependent). Our results show that only the catabolic pathway is active in these amoebae. This is in agreement with the findings that the NADPH-dependent glutamate dehydrogenase pathway is only used under low nitrogen conditions [21, 22]. In all amoebae species glutamate oxaloacetate transaminase and glutamate pyruvate transaminase were detected in the range of 275-1250 χ IO'3 IU χ mg protein'1 and 100-475 χ IO"3 IU χ mg protein"', respectively. Therefore rapid shifts in the amino acid concentrations of aspartate, glutamate and alanine within the cell can be accomplished. This should not be surprising since it is reported that free amino acids like alanine, aspartate, glutamate and glutamine play an important role in the osmotic regulation of A. castellami [12]. Glutamine synthetase activity is in the range of 30-200 χ IO"3 IU χ mg protein"1 and causes shifts between glutamate and glutamine. The specific activity of both NADH- and NADPH-dependent lactate dehydrogenase, a key enzyme in amino acid metabolism, was measured as a control for the alanine dehydrogenase assay. In A. castellami, A. polyphaga, H. vermiformis (strain Atlanta) and H. vermiformis (strain Koblenz) a specific activity of 56.9 ± 2.4 IU χ ΙΟ"3 χ mg protein"1, 151.9 ± 5.5 IU χ ΙΟ"3 χ mg protein', 80.7 ± 1.7 IU χ 10"3 χ mg protein"1 and 41.1 ± 0.7 IU χ IO"3 χ mg protein"', respectively, was measured. The NADPH-dependent lactate dehydrogenase activity was much lower: 4.2 ±0.1 IU χ IO"3 χ mg protein"', 4.3 ± 0.1 IU χ ΙΟ"3 χ mg protein"1, 0.3 ± 0.1 IU χ ΙΟ"3 χ mg protein'1 and 4.8 ± 0.2 IU χ 10"3 χ mg protein"1, respectively. NADH-dependent lactate dehydrogenase activity in the testate amoeba Netzelia tuberculata is 2-7 times higher than has been measured for the Acanthamoebid and Hartmannellid amoebae [3]. The specific activity of urate oxidase, a widely distributed enzyme in nature, has been reported for several free-living protozoan species [2, 5, 13, 15, 18, 19, 23-25]. The values for urate oxidase activity among amoebae [23-25] fall within the same range as reported in this study. For the NADPH-dependent glutamate dehydrogenase no further data are available except for Dictyostelium discoideum, where a specific activity of 13 χ IO"3 IU χ mg protein"1 was measured. This enzyme was also detected in both H. vermiformis strains (30-35 χ 10"3 IU χ mg protein"1), but appeared to be low in A. castellami and A. polyphaga (2-4 χ 10"3 IU χ mg protein'1).

61 The data for the specific activities of glutamine synthetase, both NADH- and NADPH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and NADH-dependent lactate dehydrogenase show differences between A. castellami, A. polyphaga and H. vermiformis, although no significant differences were detected between the two H. vermiformis strains. The present findings increase the knowledge of nitrogen metabolizing enzymes present in free-living soil amoeba (Fig. 1). Due to the effort of obtaining more axenic amoebae strains and novel amoebal cell culturing techniques the possibility to examine more intensively metabolic pathways in free-living amoebae will be enhanced. A complete analysis of enzyme activities of nitrogen metabolism is needed to document more fully the contribution of amoebae to the mineralization of organic nitrogen in the soil.

ACKNOWLEDGEMENTS

This research was supported by a grant from the Netherlands Integrated Program for Soil Research. We thank J.J P. Baars and MHS. Muruke for technical assistance and H.J M. Op den Camp for constructive comments.

LITERATURE CITED

1 Adam, K.M.G. 1964 The amino acid requirements of Acanthamoeba sp Neff J Protozool 11 98-100 2 Alamillo, J.M., Cárdenas J., & Pineda M. 1991 Purification and molecular properties of urate oxidase from Chlamydomonas remhardtn Biochem Biophys Acta 1076 203- 208 3 Anderson, O.R. 1988 Some enzyme activities in the testate amoebae Netzeha tuberculoid J Protozool 35 19-20 4 Byers, Τ J. 1979 Growth, reproduction and differentiation in Acanthamoeba Int Rev Cytol 61 283-338 5 Childs, CE 1973 Hartmannella culbertsoni enzymatic, ultrastructural and cytochemical characteristics of peroxisomes in a density gradient Exp Parasitol 34 44- 55

62 6 Clarholm, M. 1981 Protozoan grazing of bacteria in soil - impact and importance Microb Ecol 7 343-350 7 Clarholm, M. 1989 Effects of plant-bactenal-amoebal interactions on plant uptake of nitrogen under field conditions Biol Feriti Soils 8 373-378 8 DeVincenliis, M., Salvatore, F. & Zappia, V. 1964 Nitrogen catabolism in Amoeba proteus, studies on the identification of end-products Exptl Cell Res 35 204-206 9 De JoDckheere, J.F. 1977 Use of an axenic medium for differentiation between pathogenic and non pathogenic Naeglena fowlen isolates Appi Environ Microbiol 33 751-757 10 Dolphin, W.D. 1976 Nitrogen metabolism m Acanthamoeba castellan», amino acid consumption and composition patterns Proc Iowa Acad Sci 82 171-174 11 Ferguson, A.R. & Sims, A.P. 1971 Inactivahon in vivo of glutamme synthetase and NAD-specific glutamate dehydrogenase its role in the regulation of glutamme synthesis in yeasts J Gen Microbiol 69 423-427 12 Geoffrion, J. &. Larochelle, J. 1984 The free amino acid contribution to osmotic regulation in Acanthamoeba castellami Can J Zool 62 1954-1959 13 Gerhardt, В. 1971 Zur Lokalisation von Enzymen der Microbodies in Polytomella caeca Arch Microbiol 80 205-218 14 Griffiths, B.S. 1990 A comparison of microbial-feeding, nematodes and protozoa in the rhizosphere of different plants Biol Fend Soils 9 83-88 15 Hill, D.L. & Chambers, P. 1967 The purine and pynmidine metabolism of Tetrahymena pynformis J Cell Physiol 69 321-330 16 Kenealy, W.R., Thompson, Т.Е. Schubert, K.R. & Zeikus, J.G. 1982 Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barken and Methanobactenum thermoautotrophicum J Bactenol 150 1357-1365 17 Kidder, G.W. 1967 Nitrogen distribution, nutrition, and metabolism ρ 93-159 In Florkin, M & Scheer, Β Τ (eds ), Chemical Zoology, Vol I Protozoa, Academic Press, New York 18 Lui, N.S.T. & Roels, О Л. 1970 Nitrogen metabolism of aquatic organisms I The assimilation and formation of urea in Ochromonas malhamensis Arch Biochem Biophys 139 269-277 19 Lui, N.S.T., Roels, O.A., Trout, M.E. & Anderson, O.R. 1968 Subcellular distribution of enzymes in Ochromonas malhamensis J Protozoal 15 536-542 20 Mahler J.L. 1970 A new bacterial uncase for une acid determination Anal Biochem 38 65-84 21 Meers, J.L., Tempest, D.W. & Brown, CM. 1970 Glutamine(amide) 2-oxoglutarate aminotransferase oxido-reductase (NADP), an enzyme involved in the synthesis of glutamate by some bacteria J Gen Microbiol, 64 187-194 22 МіПіп, B.J., Lea, P.J. & Wallsgrove, R.M. 1980 The role of glutamme in ammonia assimilation and reasstmilation in plants ρ 213-234 In Mora, J & Palacios, R (eds).

63 Glutamine metabolism, enzymology and regulation, Academic Press, New York and London 23 Müller, M. & Moller, K.M. 1969 Urate oxidase and its association with perosixomes in Acanthamoeba sp Eur J Biochem 9 424-430 24 Müller, M. & Moller, M. 1969 Studies on some enzymes of purine metabolism in the amoebae Chaos chaos and Amoeba proleus Comi Rend Trav Lab Carlsberg 36 463- 497 25 Müller, M., Hogg, J.F. & de Duve, С. 1968 Distribution of tricarboxylic acid cycle enzymes and glyoxylate cycle enzymes between mitochondna and peroxisomes in Tetrahymena pynfortms J Biol Chem 243 5385-5395 26 Page, F.C. 1988 A new key to freshwater and soil gymnamoeba ρ 16-17 Culture Collection of Algae and Protozoa, Freshwater Biological Association, Ambleside, Cumbna 27 Pamula, F. & Wheldrake, J.F. 1991 Purification and properties of the NADP- dependent glutamate dehydrogenase from Dtctyosteltum discoideum Mol Cell Biochem 105 85-92 28 Tyler, B. 1978 Regulation of the assimilation of nitrogen compounds Annu Rev Biochem 47 1127-1162 29 Wachtendonk, W.J. & Soldo, A.T. 1970 Nitrogen metabolism in protozoa ρ 1-56 In Campbell, J W (ed ), Comparative biochemistry of nitrogen metabolism, Vol 1 , The invertebrates, Academic Press, London 30 Woods, L.E., Cole, C.V., Elliott, E.T., Anderson, R.V. & Coleman, D.C. 1982 Nitrogen transformations in soil as affected by bactenal-microfaunal interactions Soil Biol Biochem, 14 93-98

64

Chapter 6

Differences in Isoenzyme Patterns of Axenically and Monoxenically Grown Acanthamoeba and Hartmannella

Peter H.H. Weekers' & Johan F. De Jonckheere2

1 Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands 2 Department of Microbiology, Institute of Hygiene and Epidemiology, J. Wytsmanstraat 14, B-1050, Brussels, Belgium

(Submitted for publication)

SUMMARY

Axenically and monoxenically grown free-living soil amoebae Acanthamoeba castellana, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were studied by Polyacrylamide isoelectric focusing in the pH range 3-10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellana, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Both Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM. Isoenzymatic typing is traditionally performed with lysates from axenic cultures. Comparison of zymograms from axenically and monoxenically

67 grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

INTRODUCTION

Isoenzymatic typing of protozoans, especially Acanthamoeba [7, 13], Naegleria [5, 6, 11, 15-17], Giardia [1, 12], Vahlkampfia [4] and Entamoeba [9, 10] has proved to be a highly discriminating method for identification to the species level. It was successfully applied to the systematic identification of axenized strains isolated from various environments like fresh water, soil, industrial cooling water, chlorinated swimming pools, sewage, tap water, air conditioners and contact lens cases. Most frequently combinations of several enzymes were used for the comparison of strains and isoenzymatic typing of protozoans. For example esterase and acid phosphatase were used to divide Acanthamoeba strains into several groups which did not always correspond with the traditional species designation [2, 3, 19]. A combination of up to 15 different enzymes were used in order to determine genetic heterogeneity in natural populations of Acanthamoeba polyphaga [8], genetic variation in Giardia lamblia [12], and phylogenetic relationships in Naegleria [16]. Isoenzymatic analyses were usually performed with lysates from axenic cultures. However, in natural environments the free-living soil amoebae consume a wide variety of food, especially bacteria. Little is known about the influence of the bacterial food on typical isoenzyme patterns. Isoenzyme patterns of axenically grown amoebae may differ from those of monoxenically grown amoebae. Therefore comparison of strains by isoenzymatic typing can be affected dramatically if both axenic and xenic cultures were used together. We report on differences in isoenzyme patterns of axenically and monoxenically grown free-living amoebae, and also on differences in isoenzyme patterns of three different axenic Hartmannella vermiformis strains in comparison with Acanthamoeba castellami and A. polyphaga.

68 MATERIALS AND METHODS

Organisms and cultivations. Acanthamoeba castellami strain CCAP- 1501/1B (originally from Neff) and Acanthamoeba polyphaga strain CCAP- 1501/3C (originally strain OX-1 from Sawyer) were obtained as axenic cultures from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, Cumbria, UK). Hartmannella vermiformis strain CDC-19 (also available as ATCC-50236) was obtained as axenic culture from Dr. B.J. Fields (Respiratory Disease Branch, Center for Infectious Disease, Atlanta, USA) and H. vermiformis strain OS-101 was obtained as axenic culture from Dr. R. Michel (Emst-Rodenwaldt Insititut, Koblenz, Germany). H. vermiformis strain CCAP- 1534/7B (originally strain 197 from Page) was obtained as monoxenic culture from the Culture Collection of Algae and Protozoa (CCAP, Ambleside, Cumbria, UK). It was originally cultured in Neff Amoeba Saline medium [14] with Escherichia coli К12, but we axenized the strain. All axenically grown amoebae were cultured in PYNFH-medium (ATCC media handbook; ATCC- 1034). Amoebae from axenic cultures were harvested by centrifugation (10 min, 750 χ g) and washed (3 times) using Neff Amoeba Saline. Cells were concentrated in Neff Amoeba Saline and immediately used to inoculate the different media. E. coli K12 and Pseudomonasfluorescens wer e cultured overnight in nutrient broth (Oxoid, Basingstoke, UK) at 30°C under constant shaking. Bacterial cells were harvested by centrifugation (10-15 min, 25,000 χ g) and washed (3 times) using Neff Amoeba Saline. The bacteria were resuspended in Neff Amoeba Saline and were used as substrate in monoxenic cultures.

Axenic and monoxenic cultures. Roux flasks were used to cultivate each amoeba axenically in PYNFH-medium (pH 6.5) and monoxenically in modified Neff Amoeba Saline (20 mM phosphate, pH 6.5) with E. coli K12 or P. fluorescens as substrate. Roux flasks were inoculated with amoebae to yield an initial amoebal density of 104 cells per ml. Additionally, monoxenic cultures were inoculated with either E. coli Kl2 or P. fluorescens to yield an initial bacterial density of 109 cells per ml. As a control for autolysis of the bacteria used in the monoxenic cultures, E. coli K12 and P. fluorescens were also inoculated in Neff Amoeba Saline at the same initial cell density as the

69 monoxenic cultures but without amoebae (Table 1.). Axenic and monoxenic cultures and the controls were incubated at 30°C for several days to yield a final amoebal density of approximately 10* cells per ml.

Table 1. Axenic and monoxenic test combinations used for isoenzyme partem comparison. Identification numbers for lysates used for isoenzyme electrophoresis.

Cultures Amoebae strains8 Bacterial controls HVA HVK HVN AC AP viable lysozyme0

Axenic 1 4 7 10 13 -

on Eca 2 5 8 11 14 16 17

onPf0 3 6 9 12 15 18 19 a Abbreviations: Hartmannelia vermiformis strain CDC-19 (HVA), H. vermiformis strain OS-101 (HVK), H. vermiformis strain CCAP-1534/7B (HVN), Acanthamoeba castellami strain CCAP-1501/1B (AC), Acanthamoeba polyphaga strain CCAP-1501/3C (AP), Escherichia coli Kl 2 (Ее) and Pseudomonas fluoresceins (Pf). Controls for bacteria only. c Lysozyme treated lysates (17, 19) are obtained as described in the materials and methods.

Preparation of lysates. Axenic and monoxenic cultures and controls were harvested by centrifugation (10 min , 750 χ g) and washed (3 times) using Neff Amoeba Saline to remove the PYNFH-medium or most of the bacterial cells, respectively. Cells were concentrated by centrifugation and transferred into Eppendorf tubes. Axenic and monoxenic amoebal lysates and lysates for the bacterial controls were prepared immediately by freezing/thawing using ethanol/solid carbon dioxide. In addition, half of the bacterial cells from the controls were treated with lysozyme (12,000 IU χ ml"1, 30 min, 30°C) to yield a second set of bacterial controls. The lysates were stored at -80°C until use. The lysates were thawed on ice immediately before use.

70 Protein assay. The protein content of the lysates was determined by means of a standard protein assay with bovine γ-globulin as standard (Protein assay kit I, cat nr.: 500-0001; Bio-Rad, Veenendaal, The Netherlands).

Isoelectric focusing (IEF). Isoenzyme analysis was done by isoelectric focusing over a pH range of 3-10 (Bio-lyte 3/10, Bio-Rad, Veenendaal, The Netherlands) on 5% Polyacrylamide gels (25%T, 3%C). Isoelectric focusing was performed for two gels (100 χ 125 χ 1.6 mm each) simultaneously on a horizontal flatbed type apparatus (Bio-Rad Biophoresis System and power supply 3000ХІ). The anolyte used was 1 N H3P04 and the catholyte was 1 N NaOH, and all IEF-gels were cooled at 4°C. Prefocusing was carried out for 15 min at 500 V, 300 m A, 15 W with a constant power of 14 W. Twenty μΐ of the lysates containing approximately 100 μg protein were applied on filter paper strips (3x8 mm). Isoelectric focusing was performed using 1500 V, 300 m A and 15 W with a constant power of 14 W, for 120 min. IEF-markers (IEF markerkit, cat nr.: 07-0471-01, Pharmacia, Woerden, The Netherlands) were run simultaneously on each gel.

Developing solutions for isoenzyme staining. At the end of the run acid phosphatase (AP; orthophosphoric monoester phosphohydrolase, EC 3.1.3.2), propionyl esterase (PE; carboxylic ester hydrolase, EC 3.1.1.1), malate dehydrogenase (MDH; L-malate:NAD oxidoreductase, EC 1.1.1.37), glucose phosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), phosphoglucomutase (PGM; a-D-glucose-1,6-diphosphate:a-D-glucose-1 - phosphate phosphotransferase, EC 2.7.5.1) [6] and alcohol dehydrogenase (ADH; alcohol:NAD oxidoreductase, EC 1.1.1.1) [18] were visualized. The reproducibility and position of the isoenzyme bands was ascertained with several extracts.

RESULTS

Axenic cultures. The isoenzyme patterns for acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) of

71 the axenically grown strains were distinct between Hartmannella and Acanthamoeba. Hartmannella as well as Acanthamoeba possessed AP, PE and MDH activities but their zymograms showed different patterns (Fig. 1). For both Acanthamoeba strong bands appeared for ADH while for GPI and PGM bands were extremely weak. Differences between Acanthamoeba and Hartmannella were very explicit for ADH, GPI and PGM. Hartmannella lacked ADH but GPI and PGM activities were present. Also differences in zymograms of individual strains occurred but were smaller between the three Hartmannella vermiformis strains than between both Acanthamoeba species (Fig. 1).

Axenic versus monoxenic cultures. The isoenzyme patterns for acid phosphatase (AP), propionyl esterase (PE), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were distinct between axenically and monoxenically cultured amoebae of the same strain (Fig. 2). To obtain visible bands in the lanes for lysates of monoxenic cultures the IEF-gels for AP, PE, GPI and PGM had to be extensively overexposed to obtain clear bands. Comparison of zymograms of axenically and monoxenically grown amoebae of the same species showed a lower intensity and even lack of typical isoenzyme bands in lysates of monoxenic cultures. In addition to the absence of typical bands in isoenzyme patterns for PGM in the three Hartmannella strains grown monoxenically on Pseudomonas fluorescens(Fig . 2; lanes 3, 6 and 9), also the presence of extra bands was observed (pi 4.3). These extra bands were probably not due to enzyme activity from P. fluorescens because of differences in pi values as compared to the bacterial controls (Fig 2; lanes 18 and 19). Controls for viable bacteria (Fig. 2; lanes 16 and 18) and lysozyme treated bacteria (Fig. 2; lanes 17 and 19) showed only small differences in the intensity of their isoenzyme bands depending on the bacteria and enzyme. Most bacterial controls showed faint bands for AP, PE, GPI and PGM with exception of PE where P. fluorescens revealed very strong bands and Escherichia coli Kl2 showed no bands at all.

72 AP PE MDH

-3.5 -3.5 -3.5

-4.5 -4.5 -4.5 8 m -tî( « •i - 5.2 -5.2 -5.2 - 5.8 - 5.8 -5.8 -6.6 - -6.6 -6.6 -7.4 -7.4 -7.4

-8.2 - 8.2 -8.2 -8.6 - 8.6 I -8.6 - 9.3 ¥ M -9.3 — -9.3

12 3 4 5 12 3 4 5 12 3 4 5

ADH GPI PGM

-3.5 -3.5 -3.5

-4.5 -4.5 Ш — 4.5 -5.2 -5.2 - 5.2 -5.8 -5.8 »1 - 5.8 -6.6 -6.6 _6.6 -7.4 III -7.4 - 7.4 -8.2 -8.2 - 8.2 -8.6 -8.6 - 8.6 -9.3 -9.3 - 9.3

12 3 4 5 12 3 4 5 12 3 4 5

Fig. 1. Isoenzyme patterns for acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) of axenically cultivated Acanthamoeba and Hartmannella. The index numbers indicate the lysates of axenically grown Acanthamoeba castellami strain CCAP-1501/1B (1) and Acanthamoeba polyphaga strain CCAP-1501/3C (2), Hartmannella vermiformis strain CDC-19 (3), H. vermiformis strain OS-101 (4), H vermiformis strain CCAP-1534/7B (5). Isoelectric points (at the right) were obtained with calibration proteins.

73 AP

-3.5

-4.5 І ** t I * -5.2 -5.8 -6.6 I -7.4 • •8.2 "8.6

'-Ι-** -9.3

1 2 3 4 5 6 7 β 9 10 11 12 13 14 15 16 17 18 19

PE

m m -3.5 "4.5 - 5.2 -5.8 .6.6

'8.2 "8.6 -9.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Fig. 2. Differences in isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) for axenically and monoxenically grown Acanthamoeba castellami strain CCAP-1501/1B, Acanthamoeba polyphaga strain CCAP-1501/3C, Hartmanneila vermiformis strain CDC-19, H. vermiformis-*

74 GPI

- 3.5

4.5 5.2 5.8 6.6 I І' < 7.4 8.2 8.6 9.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

PGM

-3.5

-4.5

-5.2 • ! - 5.8 H . 6.6 / - 7.4

- 8.2 - 8.6 - 9.3 '«

6 7 8 9 10 11 12 13 14 15 16 17 18 19

—> strain OS-101, H. vermiformis strain CCAP-1534/7B and the bacterial controls. For identification of the specific test combinations and lanes on the gels see Table 1. The isoelectric points, indicated on the right of the gels, were obtained with calibration proteins.

75 DISCUSSION

Isoenzyme analysis by means of isoelectric focusing is a very useful tool for comparison and identification of protozoa in clinical and phylogenetic studies. We showed that isoenzyme analysis using AP, PE, MDH, LDH, GPI and PGM is an adequate technique to distinguish axenically cultivated Hartmannella from Acanthamoeba and to reveal differences within the species Hartmannella vermiformis. Differences in isoenzyme patterns within a species have also been reported earlier for Naegleria [11, 16] and Acanthamoeba [6, 7]. The use of isoenzyme patterns of axenically grown strains has already proven its value in the identification of many protozoa. However, isoenzyme patterns for monoxenically grown strains and comparative studies on zymograms of axenically and monoxenically grown amoebae are sparse. Our results revealed differences in typical isoenzyme patterns between lysates of axenically and monoxenically grown amoebae. In most cases the intensity of the bands was strongly reduced or bands were even absent. In some cases the presence of extra bands was observed, as shown for the Hartmannella strains with PGM (Fig. 2; lanes 3, 6 and 9). These changes in isoenzyme patterns do not agree with results previously reported for Naegleria. A lower staining intensity, although not as dramatically as observed in our experiments (Fig. 2), has also been reported for monoxenic cultures of Naegleria [17], but the bacteria had no influence on the interpretation and specificity of the isoenzymatic patterns of each strain [17]. The free-living amoebae Hartmannella and Acanthamoeba occur in various natural environments in which they mainly feed on bacteria. Differences in isoenzyme patterns, are detected when the amoebae were cultured on a bacterial substrate as compared to the typical banding patterns obtained with lysates from axenic cultures. Therefore, isoenzyme analysis for the identification of amoebae should be used with precaution when different growth conditions are used.

ACKNOWLEDGEMENTS

The authors thank Mr. A.M.W. Engelberts for his help with PAA-IEF gel

76 electrophoresis and activity assays. This research was supported by a grant from the Netherlands Integrated Program for Soil Research.

LITERATURE CITED

1 Chaudhuri P., De, Ач Bhattacharya Α., Pal S.Ch. & Das P. 1991 Identification of heterogeneity in human isolates of Giardia lamblia by isoenzymes Zbl Bakt 274 490- 490 2 Costas M. & Griffiths AJ. 1984 The esterases and acid phosphatases of Acanthamoeba (Amoebida, Acanthamoebidae) Protistologica 20 33-41 3 Dagget P-M, Brook D.R. & Nerad T.A. 1981 Relationships among 31 strams of Acanthamoeba based upon non-morphological characters J Protozoal 28 22a 4 Dagget P-M. & Nerad T.A. 1983 The biochemical identification of Vahlkampfud amoebae J Protozool 30 126-128 5 De Jonckheere J.F. 1977 Use of an axenic medium for differentiation between pathogenic and non-pathogenic Naeglena fowlen isolates Appi Environ Microbiol 33 751-757 6 De Jonckheere J.F. 1982 Isoenzyme patterns of pathogenic and non-pathogenic Naeglena spp using agarose isoelectric focusing Ann Microbiol Inst Pasteur 133 319- 342 7 De Jonckheere J.F. 1983 Isoenzyme and total protein analysis by agarose isoelectric focusing, and taxonomy of the genus Acanthamoeba J Protozool 30 701-706 8 Jacobson L.M. & Band R.N. 1987 Genetic heterogeneity in a natural population of Acanthamoeba polyphaga from soil, an isoenzyme analysis J Protozool 34 83-86 9 Mathews H.M., Moss D.M., Healy G.R. & Visvesvara G.S. 1983 Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species J Clin Microbiol 17 1009- 1012 10 Moss D.M. & Mathews H.M. 1987 A fast electrophoretic isoenzyme technique for the identification of invasive and non-invasive Entamoeba histolytica and "E histolytica- like" organisms J Protozool 34 253-255 11 Moss D.M., Brandt F.H., Mathews H.M. & Visvesvara G.S. 1988 High-resolution Polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naeglena species J Protozool 35 26-31 12 Moss D.M., Visvesvara G.S., Mathews H.M. & Ware D.A. 1992 Isoenzyme comparison of axenic Giardia lamblia strains J Protozool 39 559-564 13 Moura H„ Wallace S. & Visvesvara G.S. 1992 Acanthamoeba healyi N Sp and the isoenzyme and immunoblot profiles of Acanthamoeba spp, group 1 and 3 J Protozool 39 573-583

77 14 Page F.C. 1988 A new key to freshwater and soil gymnamoeba ρ 16-17 Culture Collection of Algae and Protozoa, Freshwater Biological Association, Ambleside, Cumbria 15 Pernio P. 1984 Isoenzyme patterns of pathogenic and non-pathogenic thermophilic Naeglena strains by isoelectric focusing M J Parásito! 14 459-465 16 Pernia P., Caríou M-L. & Jaquiers Α. 1985 Biochemical identification and phylogenetic releationships in free-living amoebas of the genus Naeglena J Protozool 32 592-603 17 Pernio P. & Grelaud G. 1989 Application of isoenzymatic typing to the identification of nonaxenic strains of Naeglena (Protozoa, Rhizopoda) Parasitol Res 75 595-598 18 Shaw C.R. & Prassad R. 1970 Starch gel electrophoresis of enzymes, a compilation of recipes Biochem Genetics 4 294-320 19 Tyndal R.Z., Willaert E., Stevens A. & Nicholson A. 1979 Pathogenic and enzymatic characteristics of Acanthamoeba isolates from cultured tumor cells Protistologtca 15 17- 22

78

Chapter 7

Bacteriolytic Activities of the Free-Living Soil Amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis

Peter H.H. Weekers, Andreas M.W. Engelberts & Godfried D. Vogels

Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands

(Submitted for publication)

ABSTRACT

Bacteriolytic activities of axenically grown free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometrical assay revealed that the specific bacteriolytic activities of both Acanthamoeba species were approximately twice as high as those of the three Hartmannella strains tested. Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus and Pseudomonas fluorescens were more easily lysed than the other bacteria tested. Agrobactermm tumefaciens and Arthrobacter simplex were hardly affected at all by the amoebal bacteriolytic activities. The three Gram-positive bacteria were more easily lysed than Gram-negative bacteria, with exception of P. fluorescens. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3-10 was performed in combination with a newly developed activity assay. The activity assay, consisting of an agar/bacteria gel-overlay, revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pi points of bacteriolytic

81 activities in Acanthamoeba and Hartmannella were shown. Bacteriolytic activities of Hartmannella were more pronounced and observed in the pi range of 4.0-9.3 while for Acanthamoeba the range was pi 4.5-8.9.

INTRODUCTION

Soil amoebae depend upon bacteriolytic enzymes to degrade the cell wall components of the microorganisms they use as a food source. Not all bacterial cell walls seem to be equally well degradable. Anionic polyelectrolytes in the bacterial cell walls such as hyaluronate, teichoic acid, branched polysaccharides, lipopolysaccharides, lipids and waxes, might be able to hamper bacteriolytic activity [9]. Lysozyme (mucopeptide N-acetylmuramoyl- hydrolase, EC 3.2.1.17) is a bacteriolytic basic protein of low molecular weight, stable at acid pH and high temperatures with the capacity to hydrolyze ß(l-»4)glycosidic linkages between N-acetylmuramic acid and N-aceryl- glucosamine of the bacterial cell wall mureins. Multiple forms of lysozyme were reported to occur in avian egg-white [1, 13] and in polymorphonuclear leukocytes of some species of birds [14] as well as in the tissues or secretions of patients suffering from leukemia [15]. Powerful bacteriolytic enzymes of the lysozyme group were found to be synthesized also by small free-living amoebae, which feed on bacteria [2-4, 8, 16, 18, 20]. In Acanthamoeba castellami the presence of two separated bacteriolytic enzymes was first reported in the early seventies [5, 6, 8]. This study reports on the bacteriolytic activity of the free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and three different strains of Hartmannella vermiformis. Bacteriolytic activities of these amoebae towards various bacteria were quantitated spectrophotometrically using kinetic assays. Isoelectric focusing gel-electrophoresis in combination with a newly developed activity assay was applied to reveal typical banding patterns for bacteriolytic activities among the amoebae and towards different bacteria.

82 MATERIALS AND METHODS

Organisms and cultivations. Acanthamoeba castellami (strain CCAP- 1501/1B) and Acanthamoeba polyphaga (strain CCAP-1501/3C) were obtained as axenic cultures from the Culture Collection of Algae and Protozoa (CCAP; Ambleside, UK). Hartmannella vermiformis strain Atlanta (strain CDC-19) was obtained as axenic culture from Dr. B.J. Fields (Respiratory Disease Branch, Center for Infectious Disease, Atlanta, GA, USA) and H. vermiformis strain Koblenz (strain OS-101) was obtained as axenic culture from Dr. R. Michel (Emst-Rodenwaldt Institut, Koblenz, Germany). H. vermiformis strain Nijmegen (strain CCAP-1534/7B) was obtained as monoxenic culture from the Culture Collection of Algae and Protozoa (CCAP, Ambleside, UK). It was originally cultured in Neff Amoeba Saline medium (AS) [17] with Escherichia coli K12, but we brought it in axenic culture. All amoebae were cultivated in PYNFH- medium as described previously [23]. Agrobacterium tumefaciens, Arthrobacter simplex, Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Escherichia coli К12, Klebsiella aerogenes, Micrococcus luteus, Pseudomonas fluorescens and Serratia marcescens were cultured overnight in nutrient broth (Oxoid, Basingstoke, UK) at 30°C on a shaking incubator.

Preparation of lysates and agar overlays. Exponentially growing amoebae cells were harvested by centrifugation (10 min , 750 χ g) and washed (3 times) with Neff Amoeba Saline [17]. Cells were concentrated by centrifugation and transferred into Eppendorf tubes in which the amoebal lysates were prepared immediately by freezing/thawing using ethanol/solid carbon dioxide. The lysates were stored at -80°C until use. Immediately before use, amoebal lysates were thawed on ice. Bacterial cells were harvested by centrifugation (10-15 min, 25,000 χ g) and washed (3 times) using Neff Amoeba Saline [17]. Bacterial cells were concentrated by centrifugation, transferred into Eppendorf tubes, immediately lyophilized and stored at -20°C until use. Bacterial cells used for the spectrophotometrical activity assay and the agar overlay of the isoelectric focusing gels (IEF) were resuspended in a citric acid-sodium citrate buffer (20 mM, pH 5.0). The agar overlay for the IEF-gel was prepared by melting 0.45 g ultrapure agar (Difco, Detroit, MI,

83 USA) in 13 ml citric acid-sodium citrate buffer (20 mM, pH 5.0). The molten agar was cooled down to 60-65°C and 2 ml of a bacterial cell suspension

(OD550 = 1.0 if diluted 100 times) was added and mixed thoroughly. The bacteria/agar suspensions were poured into casting trays (100 χ 125 χ 0.8 mm) and were immediately, after the agar had solidified, used to cover the IEF-gel.

Bacteriolytic activity assay. The amoebal lysates were diluted with citric acid-sodium citrate buffer (20 mM, pH 5.0, 4°C) immediately before use to a concentration of 100 μg protein per ml. A sample (1.5 ml) was placed in a temperature (45°C) controlled cuvette of a kinetic spectrophotometer (Diode Array, Hewlett Packard). A time scan was started to record the optical density (550 nm) of the amoebal lysate and to assure stable initial conditions. After 25 sec the bacteriolytic assay was started by adding 15 μΐ of the bacterial cell suspension to yield an OD550 of approximately 1.0. Decrease of the ODJ50 was recorded during 10 min (software: HP 89531A MS-DOS UV/VIS Operating System, Hewlett Packard). Kinetic data were calculated from the values of the initial slope in the assay and specific bacteriolytic activities are expressed as Δ 1 1 OD550 χ mg amoebae protein" χ min" .

Isoelectric focusing and bacteriolytic activity assay. Bacteriolytic activities were visualized with enzymes separated by isoelectric focusing over a pH range of 3-10 (Bio-lyte 3/10, Bio-Rad, Veenendaal, The Netherlands) on homogeneous 5% Polyacrylamide gels (25%T, 3%C). Isoelectric focusing was performed for two gels (100 χ 125 χ 1.6 mm each) simultaneously on a horizontal flatbed type apparatus (Bio-Rad Biophoresis System and Power Supply ЗОООХі). Prefocusing was carried out for 15 min at 500 V, 300 m A, 15 W with a constant power of 14 W. Samples of the amoebal lysates (20 μΐ) were applied on filter paper strips (3x8 mm), each containing approximately 100 μg amoebal protein. Migration was performed using 1500 V, 300 mA and 15 W with a constant power of 14 W for 120 min. The anolyte used was 1 N

H3P04 and the catholyte was 1 N NaOH. All IEF-gels were cooled on a 4°C platform. The isoelectric points (pi) of the isoenzyme bands were extrapolated from plots of IEF-markers (IEF markerkit, cat nr.: 07-0471-01, Pharmacia, Woerden, The Netherlands) with known pi versus the distance of their electrophoretic migration from the cathode. Immediately after isoelectric

84 focusing the gels were covered with agar/bacteria overlays as described above and incubated at 45°C. To reveal the bacteriolytic patterns pictures were taken after 4 and 21 h of incubation using a dark field photography technique.

Protein assay. The protein content of the amoebal lysates and the bacteria were determined by means of a standard protein assay with bovine γ-globulin as the standard (Protein assay kit I, cat nr.: 500-0001; Bio-Rad, Veenendaal, The Netherlands).

RESULTS

Bacteriolytic activity. The specific bacteriolytic activities of cell lysates of Acanthamoeba castellami, Acanthamoeba polyphaga and three different strains of Hartmannella vermiformis on ten bacterial suspensions are presented in Table 1.

Table 1. Specific bacteriolytic activities* of amoebal lysates on various bacterial suspensions.

В act en a A castellami A polyphaga H vermiformis Atlanta Koblenz Nijmegen

A tumefaciens 0 003 0 022 0 223 0 164 0 003 A simplex 0 423 0 421 0 129 0 035 0 003 В megatenum 0 840 0 834 0415 0 134 0 075 В sub tilt s 1 326 1422 0 606 0 583 0 022 С vinosum 2 634 2 790 0 595 0 368 0 476 E coli 0 178 0119 0251 0 094 0 158 К aerogene s 0 040 0 052 0 029 0 006 0 024 M luteus 2 136 3 174 1080 0 303 0 262 Ρ fluorescens 15 780 15 000 9 960 5 580 6 480 S marce scens 0 024 0016 0 006 0013 0015

Specific bacteriolytic activities are expressed as Δ OD550 χ mg amoebal protein' χ mm'

85 All amoebal lysates were active but the specific bacteriolytic activity of a single amoebal lysate varied strongly (over 103-times) for different bacteria. Bacillus megatermm, Bacillus subtilis, Chromatmm vinosum. Micrococcus luteus and Pseudomonas fluoresceins were most easily lysed. Serratia marcescens and Klebsiella aerogenes were hardly affected, while Arthrobacter simplex, Agrobactenum tumefaciens and Escherichia colt К12 were lysed at various rates. Specific bacteriolytic activities among amoebal lysates varied considerably (10- to 100-fold) for a single bacterial substrate. The bacteriolytic activities of A. castellami and A. polyphaga were higher than those of the H. vermiformis strains. Differences in specific bacteriolytic activities between A. castellami and A. polyphaga and, between the three different H. vermiformis strains are rather small in most instances. The specific bacteriolytic activities of Acanthamoeba and Hartmannella have their highest value at pH 5.0 and 5 5, respectively (Fig. 1A), and possess a broad temperature range with an optimum between 40-55°C (Fig. IB).

Bacteriolytic isoenzymes patterns. Pictures of the isoenzyme patterns of the bacteriolytic activities on PAA-IEF gels АэгЛ. castellami, A. polyphaga and H. vermiformis (strain Atlanta and Koblenz) are shown in Fig. 2. The amoebal lysates revealed typical bacteriolytic lysis patterns in the agar overlay containing the different bacteria. B. subtilis, C. vmosum, M. luteus and P. fluorescens were affected most strongly by all amoebal lysates, showing the most intense lysis patterns. A. simplex and A. tumefaciens showed hardly any lysis at all, while the lysis patterns of B. megatermm, E. colt К12, К. aerogenes, and S. marcescens varied depending on the amoebal lysate used. Typical lysis bands for Acanthamoeba appeared after a relative short time of incubation (2-4 h) at pi 4.70, 5.80/5.90, 6 55/6 70, 7.35, 8.40 and 8.90 (Figs. 2, 3). Small differences were found in bacteriolytic patterns between A. castellami and A. polyphaga. The lysis bands of both Acanthamoeba were focused around narrow pi values and were more pronounced than those of Hartmannella. Most of the bacteriolytic activities of both Hartmannella strains tested were found in four typical bands, around pi 4.00, 4 25, 4.50 and 9.30 (Figs. 2, 3) and were only well visible after a relatively long time of incubation (4-12 h). All Hartmannella bands have a broad pi range due to the longer incubation time which effects diffusion of activities in the agar/bacteria overlay on the IEF-gels.

86 30 D A castellami I 25 Ο H vermiformis (Atlanta) A χ ,-G.^ V g 2.0 ü 3 Φ ^" о α α. 1 5 D CD -Ό— E О -4 Ю in ^ 8 05 ' o~—° <3 00 30 40 5.0 60 70 pH

30 π A castellami ç О н vermiformis (Atlanta) E 25

20 g Q. 1 5 CD E χ 1 0 о in ю Q 05 О 00 10 20 30 40 50 60 70 80 temperature ( °С )

Fig. 1. Typical pH (A) and temperature (B) optima for bacteriolytic activities in Acanthamoeba castellami and Hartmannella vermiformis (strain Atlanta) lysates directed towards a Micrococcus luteus suspension.

87 3.5

4.5 -

5.2 - 5.8 -

6.6 -

7.4 -¡

8.2 - 8.6 -l

- 9.3 -.

88 H

- 3.5 - m 3.5

4.5 - - 4.5 -

5.2 - - 5.2 - 5.8 - " - 5.8 -

6.6 - f - 6.6 -ì

7.4 - - 7.4 -

8.2 - • 8.2 - - 8.6 - 8.6 - *

9.3 - • - 9.3 H

12 3 4 1 2 3 4

Fig. 2. Isoenzyme patterns for bacteriolytic activities in Acanthamoeba and Hartmannella. Figs. Α-J represent the activity tests on the PAA-IEF gels with Agrobacterium tumefaciens, Arthrobacter simplex, Bacillus megalerium, Bacillus sublilis, Chromatium vinosum, Escherichia coli К12, Klebsiella aerogenes, Micrococcus luteus, Pseudomonas fluorescens and Serratia marcescens, respectively. The index numbers 1-4 indicate the lysates of Acanthamoeba castellana, Acanthamoeba polyphaga, Hartmannella vermiformis (strain Atlanta) and H. vermiformis (strain Koblenz), respectively. The isoelectric points, indicated between the gels, were obtained with calibration proteins. Pictures were taken after 4 hours of incubation using a dark field photography technique.

89 a b с d

^4.00 4.50^ - 4.25 ^4.50 4.70^

5.80_ 5.90-^ 6.55^ 6.70^ 7.35 -

8.40 - 8.90 - -9.30

Fig. 3. Comparison of typical bacteriolytic isoenzyme patterns of Acanthamoeba and Hartmannella. Diagrammatic representation of the most pronounced lytic bands in Acanthamoeba castellami (a), Acanthamoeba polyphaga (b), Hartmannella vermiformis strain Atlanta (c) and H. vermiformis strain Koblenz (d) is given. Isoelectric points (pi) are indicated on the left and right.

DISCUSSION

The overall bacteriolytic activities in lysates of Acanthamoeba and Hartmannella could be determined by a spectrophotometrical assay. A combination of Polyacrylamide isoelectric focusing and agar/bacteria overlays revealed isoenzymatic profiles which were used to identify the most prominent

90 bacteriolytic activities by pi value. Both techniques were able to distinguish between Acanthamoeba and Hartmannella species based upon their bacteriolytic activities towards various bacterial suspensions. Acanthamoeba and Hartmannella are able to lyse Gram-positive as well as Gram-negative bacteria, but distinct differences between the sensitivity of various bacterial cells occur. The Gram-positive Bacillus megaterium, Bacillus subtilis and Micrococcus luteus were lysed at higher rates than most Gram- negative bacteria, with exception of Pseudomonas fluorescens and Chromatium vinosum. The higher rates for lysis of Gram-positive bacteria by lysates of Acanthamoeba castellami conform to previous results [6]; also high rates for lysis of P. fluorescens were reported earlier [4]. Differences between Gram- positive and Gram-negative bacteria were probably due to a better accessibility of the peptidoglycan layer in the Gram-positive bacterial cell walls. Differences in bacteriolytic sensitivities between various Gram-negative bacteria were probably effected by the presence of specific proteins, lipoproteins or lipopolysaccharides in the bacterial cell wall. These anionic polyelectrolytes located in the outer wall layer of Gram-negative bacteria are able to hamper bacteriolysis, as reported previously [9]. The differences in pH and temperature optima for the bacteriolytic activities between Acanthamoeba and Hartmannella were small, which is in agreement with previous studies on A. castellana and Hartmannella glebae [3, 6, 12, 20, 21]. Comparison of specific activities (Tab. 1) and typical isoenzyme patterns (Figs. 2, 3) showed a strong resemblance between the different Hartmannella strains. Although not as prominent as for the Hartmannella strains, also the specific activities and isoenzyme patterns for both Acanthamoeba species showed a resemblance. However, the two genera showed distinct differences. Differences in isoenzyme patterns suggest different forms of bacteriolytic activities. In earlier studies two forms of bacteriolytic endo-N- acetylmuramidase, differing in electrophoretic mobility, were discovered in amoebal cells [3-7, 18, 22]. Besides N-acetylmuramidase, other enzymes are active against mucopeptides [18], while previous findings also report on lysosomal hydrolases existing in several molecular forms [10, 11, 19]. Whether there are different bacteriolytic enzymes or only different molecular forms of a bacteriolytic enzyme present in the amoebae tested is still a question to be solved.

91 ACKNOWLEDGEMENTS

The authors thank Mr. J.P.H. Wijen for his help in developing the agar/bacteria overlay method and Mr. H.J.M. Spruyt for his contribution in the dark field photography technique. This research was supported by a grant from the Netherlands Integrated Program for Soil Research.

LITERATURE CITED

1 Arnheim, N., Hindenburg, Α., Begg, G.S. & Morgan, F.J. 1973 Multiple genes for lysozyme in birds J Biol Chem 248 8036-8042 2 Berger, D. & Mannheim, W. 1970 Anreicherung und Charactensierung eines bakteriolytischen Amobenenzyms Ζ Naturforsh 256 1002-1010 3 Drozanski, W. 1969 Bacteriolytic enzyme produced by Acanthamoeba castellami Acta Microbiol Polon 18 155-168 4 Drozanski, W. 1969 Bacteriolytic spectrum of the enzyme produced by Acanthamoeba castellami Acta Microbiol Polon 18 169-174 5 Drozanski, W. 1972 Enzymatic mode of action of the bacteriolytic enzyme from Acanthamoeba castellami Acta Microbiol Polon 21 53-62 6 Drozanski, W. 1972 Purification and properties of bacteriolytic enzyme produced by Acanthamoeba castellami Acta Microbiol Polon 2133-52 7 Drozanski, W. 1978 Two forms of bacteriolytic endo-N-acetylmuramidase in Acanthamoeba castellami Acta Biochimica Polon 25 229-238 8 Drozanski, W. 1978 Activity and distribution of bacteriolytic N-acetyl-muramidase during growth of Acanthamoeba castellami in axenic culture Acta Microbiol Polon 27 243-256 9 Ginsburg, I. 1988 The biochemistry of bacteriolysis paradoxes, facts and myths Microbiol Sci 5 137-142 10 Goldstone, Α. & Koenig, Η. 1974 Autolysis of glycoproteins in rat kidney lysosomes in vitro. Effects on the isoelectric focusing behaviour of glycoproteins, arylsulphatase, and ß-glucuronidase Biochem У 141 527-535 11 Goldstone, Α. & Koenig, Η. 1974 Synthesis and turnover of lysosomal glycoproteins. Relation to the molecular heterogeneity of lysosomal enzymes FEBS letters 39 176- 181 12 Hemelt, D.M., Mares, B. & Upadhyay, J.M. 1979 Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae Appi Environ Microbiol 38 373-378

92 13 Hermann, J4 Jolies, J. & Jollès, P. 1971 Multiple forms of duck-egg-white lysozyme - Primary structure of two duck lysozymes Eur J Biochem 24 12-17 14 Hindenburg, Α., Spitznagel, J. & Arnheim, N. 1974 Isoenzymes of lysozyme in leukocytes and egg-white evidence for the species-specific control of egg-white lysozyme synthesis Proc Natl Acad Set USA 71 1653-1657

15 Jolles, Рч Bernier, I., Berthou, J., Charlemange, D., Faure, A4 Hermann, J4 Jollès, J., Pérín, J.P. & Saint-Blanchard, J. 1974 From lysozyme to chitinases, Structural, kinetic and chrystallographic studies ρ 31-54 In Osserman, EF, Canfield, RE & Beychok, S (eds ), Lysozyme, Academic Press Ine , New York 16 Katz, W., Berger, D. & Martin, H.H. 1971 Action of an N-acetylmuramidase from a hmax amoeba on the murein of Proteus mirabilis Biochim Biophys Ada 244 47-57 17 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae ρ 16-17 Culture Collection of Algae and Protozoa, Freshwater Biological Association, Ambleside, Cumbria 18 Rosenthal, S., Reed, E J. & Weisman, R.A. 1969 Effect of lytic enzymes of Acanthamoeba castellami on bacterial cell walls J Bactenol 98 182-189 19 Stahl, P., Six, H. , Rodman, J.S., Schlesinger, P., Tulsiani, D.R.P. & Touster, O. 1976 Evidence for specific recognition sites mediating clearance of lysosomal enzymes in vivo Proc Natl Acad Sci USA 73 4045-4049 20 Upadhyay, J.M. 1968 Growth and bacteriolytic activity of a soil amoeba, Hartmannella glebae J Bactenol 95 771-774 21 Upadhyay, J.M. & Difulco, T.J. 1972 Isolation, purification, and properties of a second bacteriolytic enzyme from Hartmannella glebae Arch Biochem Biophys 149 470-475 22 Upadhyay, J.M., Mares, B.A., Hemelt, D.M. & Rivet, P.G. 1977 Purification and specificity of bacteriolytic enzyme I from Hartmannella glebae Appi Environ Microbiol 33 1-5 23 Weekers, P.H.H. and van der Drift, C. 1993 Nitrogen metabolizing enzyme activities in the free-living soil amoebae Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis J Euk Microbiol 40:251-254

93

Chapter 8

Small-Subunit Ribosomal RNA Analysis of Three Hartmannella vermiformis Strains. Sequences, Secondary Structure and Phylogenetic Implications

Peter H.H. Weekers1, Rebecca J. Gast2 & Thomas J. Byers2

' Department of Microbiology, Faculty of Science, University of Nijmegen, Toemooiveld, NL-6525 ED Nijmegen, The Netherlands 2 Department of Molecular Genetics, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA

(Submitted for publication)

ABSTRACT

Nucleotide sequences have been determined for nuclear small-subunit ribosomal RNA genes (srRNA) from three strains of the free-living Umax amoeba Hartmannella vermiformis isolated from the United Kingdom, Germany and the United States. The gene is 1,840 bp long. The average GC content is 49.6%. Sequence differences among the strains resulted from base substitutions only and ranged from 0.38-0.45%. Twelve variable sites were found and these occurred within regions of the srRNA that are known to vary in other organisms. A model for the secondary structure of the srRNA is presented and it is observed that the sequence variations among strains have negligible effects on the predicted structure. Parsimony, distance and bootstrap analyses were used to examine phylogenetic relationships between the srRNA sequences of H. vermiformis and other higher and lower eukaryotes. Of those tested, the sequences of the amoeba

95 Acanthamoeba and the alga Cryptomonas were the closest to those of Hartmannella.

INTRODUCTION

Free-living amoebae such as Hartmannella and Acanthamoeba in the Class Lobosea and Naegleria in the Class Heterolobosea [21] are found widely in nature and are both ecologically and medically important. Hartmannella and Acanthamoeba play important roles in nitrogen mineralization in soils [6, 7, 11]. Hartmannella is a possible vector for the bacterium Legionella pneumophila which causes Legionnaires disease [12, 28]. Acanthamoeba and Naegleria are opportunistic human pathogens causing eye and/or brain disease [1, 19, 28]. Persistent problems with classification of these organisms has stimulated interest in using molecular methods to reexamine phylogenetic relationships within and among genera of amoebae as a way to evaluate and refîne current taxonomie schemes. Comparison of small-subunit ribosomal RNA (srRNA) sequences has been a useful approach to evaluate phylogenetic relationships among a variety of protists [17, 25, 26] and is used here to study evolutionary relationships of the genus Hartmannella. Acanthamoeba is the only genus of lobosea for which published information on srRNA based phylogenetic relationships is available [14, 17, 26]. These studies suggested that Acanthamoeba is only very distantly related to the amoeboflagellate Naegleria and is more closely related to humans, green algae and land plants than to many other protozoans [2, 25, 26]. We have amplified, sequenced and compared nuclear srDNAs from three strains of Hartmannella vermiformis, one isolated from the United Kingdom, one from Germany and one from the United States. We use these sequences to explore the phylogenetic relationships of Hartmannella and find that it is most closely related to Acanthamoeba and the alga Cryptomonas. In addition, we examine the regions of variable sequence and their potential impact on secondary structure of the srRNA.

96 MATERIALS AND METHODS

Organisms and cultivation. Hartmannella vermiformis Strain Nijmegen, isolated in the UK, was obtained as a monoxenic culture from the Culture Collection of Algae and Protozoa (Ambleside, Cumbria, UK; strain CCAP- 1534/7B) and was cultured axenically in Nijmegen by PHHW. H. vermiformis Strain Koblenz, isolated in Germany, was obtained as an axenic culture from Dr. R. Michel (Emst-Rodenwaldt Institut, Koblenz, Germany; strain OS-101). H. vermiformis Strain Atlanta, isolated in the USA, was obtained as an axenic culture from Dr. B.S. Fields (Centers for Disease Control, Atlanta, GA, USA; strain CDC-19 [essentially the same as American Type Culture Collection #50237]). All amoebae were cultivated as static cultures at 30°C in 25 cm2 tissue culture flasks (Coming Glass Works, Coming, NY, USA) with 10 ml PYNFH medium (ATCC 1034).

DNA isolation and PCR amplification. Late log phase cultures were harvested by centrifugation at 2,000 χ g and -10' amoebae were resuspended and vortexed in 1.0 ml lysis buffer containing 200 μg/ml Proteinase K, 0.2% SDS, 10 raM Tris (pH 7.4), 10 mM NaCl and 10 mM EDTA [4]. The mixture was extracted once with 1.0 ml phenol and once with 1.0 ml chloroform/isoamyl alcohol (25:1 v/v). After precipitation with an equal volume of ethanol at -80°C, the nucleic acids were recovered by centrifugation at 16,000 χ g for 15 minutes and then resuspended to a final concentration of approximately 2-4 μg DNA per μΐ in double-distilled water. The DNA was stored at -20°C. Eukaryotic primers were used to PCR amplify complete srRNA genes as well as subfragments (Table 1). PCR conditions were: 1 min at 95°C for denaturation, 2 min at 42°C for annealing, 3 min at 72°C for extension in a run of 35 cycles. PCR products were band isolated on an 0.8% agarose gel and subsequently purified with the GeneCleanTm kit (Bio 101 Ine, Lajolla, CA, USA). All samples were stored at -20°C.

Sequencing. The full length srDNA was sequenced in triplicate in 5'->3' and 3'->5' directions using the specific primers and with the products of PCR amplified srDNA subfragments as templates. All sequencing was done with the dideoxynucleotide chain termination method [22] using the Double Stranded

97 DNA Sequencing Kit (BRL Life Technologies Inc. Gaithersburg, MD, USA). Reactions were run on 6% acrylamide-urea sequencing gels, dried, and exposed to Kodak XRP-1 diagnostic film at room temperature. Complete overlapping sequence was obtained for both strands of the DNA.

Table 1. PCR primer sequences used for sequencing of srDNA.

Primer Sequence Position in H. vermiformis gene (bp)

5'->3' directed PCR primers1: SSU1 CCGCGGCCGCGTCGACTGGTTGATCCTGCCAGTAG -11-23 373C GATTCCGGAGAGGGAGCCTGA 391-411 570C GTAATTCCAGCTCCAATAGC 593-612 PCR2 GA ACTTA A AGGA ATTGA 1152-1168 1262C GTGGTGCATGGCCGTTCTTA 1288-1307 1200C CAGGTCTGTGATGCCC 1465-1480

3'->5' directed PCR primers': 170 GCATGTATTAGCTCTAGA 158-175 373 TCAGGCTCCCTCTCCGGAATC 391-411 570 GCTATTGGAGCTGGAATTAC 593-612 1137 GTGCCCTTCCGTCAAT 1164-1179 1262 GAACGGCCATGCACCAC 1288-1304 1200RE GGGCATCACAGACCTG 1465-1480 SSU2 CCGCGGCCGCGGATCCTGATCCCTCCGCAGGTTCAC 1818-1853

1 All pnmer sequences are written 5'—>3' Underlined regions in SSU1 and SSU2 identify customized Bam Ш and Sal I sites for use in cloning

Sequence analysis. The srRNA sequences for Hartmannella strains were inferred from srDNA sequences and were used for secondary structure and phylogenetic analyses. Computation and drawing of the srRNA secondary structure model for the Nijmegen strain was done for us by the laboratory of R.

98 De Wachter using a comparative sequence analysis approach [9]. Examination of possible variations in the secondary structure of individual stem/loop regions due to sequence variations was done in our labs using Zucker's PCFOLD program, version 4.0, for RNA secondary structure prediction [27, 29] and MOLECULE, the implemented energy model and drawing program [18]. Sequences were aligned by eye based on published primary and secondary structure alignments [9, 20] using the Eyeball Sequence Editor (ESEE) [5]. The following srRNA sequences, with Genbank accession numbers in parentheses, were used: Acanthamoeba castellami Neff (M13435), Cryptomonas phi (X57162), Oryza sativa (rice) (X00755), Zea mays (com) (K02202), Chlamydomonas reinhardtii (M32703), Homo sapiens (X03205), Saccharomyces cerevisiae (M27607), and Ochromonas dánica (M32704). An initial global analysis of higher and lower eukaryotic organisms placed Hartmannella close to Acanthamoeba. Thus, further tests focused on this relationship. We examined: a) a set of nine organisms including Hartmannella, seven organisms previously identified as relatively closely related to Acanthamoeba [10, 16, 26], and Ochromonas as the outgroup, and b) a subset including the three strains of Hartmannella vermiformis, their two closest relatives, and Chlamydomonas as the outgroup. The analyses in these two cases were based on 190 and 217 informative sites out of 1381 and 1650 total aligned sites, respectively. All phylogenetic analyses used the following programs from the PHYLIP 3.4 package [13]: DNAPARS (parsimony), DNADIST (distance), NEIGHBOR (neighbor joining), DNAINVAR (Lake's and Cavender's phylogenetic invariants), and DNABOOT (bootstrapping).

RESULTS

srDNA sequences and secondary structure predictions. The complete srDNA sequences of Hartmannella vermiformis strains Nijmegen, Koblenz and Atlanta were PCR amplified using primers SSU1 and SSU2 which are complementary to the very strongly conserved 5' and 3' ends of eukaryotic srRNA genes. We obtained unambiguous srDNA sequences for all three strains (Fig. 1). The coding sequence is 1,840 bp and the average GC content is 48.59 ± 0.09%.

99 PP S o o o o o О 9 0 о α о о о о eoo Φ in о ρ О m іл m Сое vtmm ö 5 S 4 Ч >f ??s §§g SSS |§§ ggg gg§ ¡ Ο Ο Ν Ν Ν Π Μ η ЧІЛІП -«44) « β 3 β Β 4

100 Since only PCR amplification products were examined, the terminal sequences that we determined were those of the primers SSU1 and SSU2 (minus the customized restriction sites). The actual terminal sequences are unknown, but should be identical or nearly identical to those of the primers. Sequence differences among srDNAs from the three H. vermiformis strains range from 0.38-0.49% (Table 2). The predicted secondary structure of the Nijmegen strain (Fig. 2) resembles that of the Neff strain of Acanthamoeba [9, 14]. Significant differences in Hartmannella include the absence of regions E21-3, E21-4 and E43-1 and shorter lengths for the six stem/loop regions where sequence variation occurs among the Hartmannella strains.

Table 2. Percentage sequence differences and structural distances based on srDNA comparisons of three Hartmannella strains and their closest relatives.

Hv-N Hv-K Hv-A Ас-N Cph Cre

Hartmannella (Nijmegen) 0 43 0 49 35 45 22 28 24 18 Hartmannella (Koblenz) 0 0018 0 38 35 45 22 28 24 24 Hartmannella (Atlanta) 0 0030 0 0024 35 45 22 45 24 24 Acanthamoeba (Neff) 0 1503 0 1497 0 1512 34 71 37 10 Cryptomonas phi 0 1701 0 1710 0 1733 0 1347 20 55 Chlamydomonas remhardu 0 1913 0 1914 0 1922 0 1658 0 1512

Values above the diagonal are % sequence differences and those below are structural distances (substitutions/site) The dataset for distance calculations included 271 informative sites out of a total of 1,650 unambiguously aligned sites

«- Fig. 1. Sequences of the small-subunit ribosomal RNA region of Hartmannella vermiformisribosomal DNA . Underlined sequences at the 5' and 3' ends indicate SSU1 and SSU2 primer sequences; the actual sequences have not been determined in these regions. Solid symbols indicate variable positions that do (O) or do not (·) affect secondary structure.

101 Sequence heterogeneity was due to base substitutions rather than insertions or deletions and was found in the nine variable regions at twelve nucleotide positions: stem 11 (base #287), E21-1 (#665, 694), E21-2 (#714, 750, 752), E21-5 (#729), 44 (#1545), and 47 (#1730, 1732, 1734, 1756) (Figs. 1, 2). Analysis of the predicted secondary structure in the variable regions indicated that only the variation at position 729, а С in Strain Nijmegen and a G in Strains Koblenz and Atlanta, would affect secondary structure. The change from С to G would simply increase the size of the terminal loop predicted for E21-5 by one base pair.

Phylogenetic analyses. Parsimony analyses were used to explore the placement of Hartmannella srRNA sequences in: 1) a global srRNA tree, 2) a tree including a more limited group of eukaryotes, and 3) a tree limited to close relatives. The global tree is not shown, but the branch order was similar to previously published trees [2, 10, 16, 25, 26]. A single most parsimonious tree was obtained for each of the other two analyses (Figs. ЗА, 3B). The two trees are topologically consistent with each other. Bootstrap values >90% (Fig. ЗА) strongly support the branching order of Chlamydomonas reinhardtii, Zea mays and Oryza sativa, and the branch including Saccharomyces cerevisiae and Homo sapiens, but values <75% offer only limited support for the branching order of Cryptomonas phi, Hartmannella vermiformis and Acanthamoeba castellami. Stronger support for the latter branches was obtained by restricting the analysis to the two amoebae, Cryptomonas, and the outgroup Chlamydomonas. In this case, the branch linking the amoebae had a bootstrap value of 94% (Fig. 3B).

Fig. 2. Secondary structure model for srRNA of Hartmannella vermiformis (Nijmegen). The 5'-terminus is indicated by a dot, the З'-terminus by an arrowhead. Helices are numbered in the order of occurrence from 5'- to З'- terminus. Helices bearing a composite number preceded by E are specific and display extreme length variation among eukaryotes [9]. Numbers in parentheses indicate positions that vary in the 3 strains. Although not indicated, position 752 also is variable. —>

102 „ц и · ·"· β .υ , α • cea аде есілідл—ccuCu** ддие*свсвАдс':оа ес чс ». oeu, _uuB овдвии оододии. uc*e OCaCuuO ueoon.» 39 • ï ΐ .* î * ue **

e*** .» с ï» * u "•О Ч se s; Д42 ; s» с с 23 ï / eecc и с 35' V S? ». v 24 °ч\ ss •e·. Cv V™ ". uW С O С II Z' · c 'o .c o » u 0»o u « с c OC ¡> μ 25'· t SS 34»; в с u4o £ ІЛ Α*· ee '•i i'° β u \ (à U о и "*'! s"' (IMS) E21-8 E21-B Д С 0 с *· •" «. .· V" 2β .. гбс"-.и ^>Л> u* V" „" OQCUMABUAAUOOJ « „0« „* Ü це 4с "." Чс"33 32^ auuocuuuauucueocuuc S' V·*"* β в E21 7 *· IMO ιι.,ιικιι u*·"" ч ди**иОО»ді,д » c υ caccu Si \ a/': зі ° L· 20Î .· . .» •с ч л _.45. E2i-6. ;>o-e" »-·: r q β 1 :г«вод, иодсвлдооосд «всасадои ' (1731) ._."«"·..· *ÏE2E211 22 ..S («J) t."4 д ,-адссоисси дсивииисссои исасасиса с ; Ч ', •°46 -J г t ï »«»„.* е 0™>) (1734) / E21 5 (714) '"•" Ч г : з' ста») с. \ I * св °о « в с иссилсс иивдасааи во ОЯЛС *с<иои«»ео*" "* γ с л »

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103 Hartmannella Oryza sativa vermiformis (Nijmegen) Zea mays Acanthamoeba castellami (Neff)

Chlamydomonas reinhardtii Cryptomonas phi

Saccharomyces ' Ochromonas dánica cerevisiae Homo sapiens

Hartmannella В vermiformis (Nijmegen) Hartmannella vermiformis Hartmannella (Atlanta) vermiformis \ (Koblenz) 100

94

Acanthamoeba 100 castellami (Neff) ' Cryptomonas phi

Chlamydomonas reinhardtii

Fig. 3. Parsimony trees showing phylogenetic relationships of H vermiformis inferred from srRNA sequences Phylogenetic relationships of H vermiformis to A) representative eukaryotes and, B) its closest relatives Trees are unrooted most parsimonious trees Numbers at nodes are the frequency (%) at which a cluster appeared in a bootstrap test of 100 runs

•\r\A Trees constructed from neighbor-joining analyses (DNADIST) with distance values from the Kimura 2-parameter analysis or the Jukes-Cantor analysis, had the same topology as the parsimony trees (not shown). Distances calculated for the three strains of H. vermiformis using DNADIST and Kimura's 2-parameter test indicate that they differ from each other by a maximum of 0.003 substitutions per site, whereas, they differ from Acanthamoeba, Cryptomonas, and Chlamydonomas by 0.15, 0.17, and 0.19 substitutions per site, respectively (Table 2). Although distance values (Table 2) suggest that Hartmannella is related closer to Acanthamoeba than to Cryptomonas and that Acanthamoeba is closer to Cryptomonas than to Hartmannella, the distances are small. Bootstrap and Lake's and Cavender's phylogenetic invariants tests were used to determine if a statistically significant branching order among the three strains of Hartmannella could be identified, but the results were inconclusive. This was true whether the data used included Hartmannella and its closest relatives Acanthamoeba and Cryptomonas or were limited to the two amoeba genera and used the 12 variable positions in the Hartmannella sequences and the corresponding positions in the Acanthamoeba sequence.

DISCUSSION

Sequence variation and secondary structure. The srRNA secondary structure model proposed for Hartmannella vermiformis and the locations of variable nucleotides are consistent with models for other eukaryotic srRNAs [9, 15, 20]. The Hartmannella secondary structure model includes typical eukaryotic secondary structure elements. All variable sites are within regions found to be variable in other eukaryotic srRNAs [9]. E21-3, E21-4 and E43-1, three variable regions present in srRNA from Acanthamoeba [9], are absent from the srRNA of Hartmannella and a number of stems present in both amoebas are shorter in Hartmannella. No Hartmannella variable regions are longer than in Acanthamoeba and no variable regions occur in Hartmannella that are not also present in Acanthamoeba. These differences between the two genera are consistent with the fact that the 1,840 bp Hartmannella srRNA is 463 bases shorter than the 2,303 bp Acanthamoeba srRNA [14].

105 Intraspecific sequence differences among the three strains of H. vermiformis is less than 0.5% even though one strain is from North America and the others from Europe (Table 2). A sequence determined by J.H. Gunderson and S.J. Goss for a fourth strain of H. vermiformis (ATCC #30966 and Genbank Accession #M95168) differed from our Atlanta strain by 0.3%. These differences are lower than srRNA sequence differences of 1.6-3.8% for Acanthamoeba castellami [R.J. Gast, D.R. Ledee and T.J. Byers, in preparation]. The latter higher values may simply reflect a greater uncertainty in the classification of Acanthamoeba. This possibility is consistent with the observation of Gast et al. that intraspecific and interspecific differences were comparable in Group II species of this genus. It is hoped that further analysis of srRNA sequences from Hartmannella and other amoeba genera will provide a molecular basis for defining amoeba taxa based on phylogenetic relationships as well as a useful measure of the validity of current amoeba taxa.

Phylogeny. The tree topology that we found (Fig. ЗА) is in general agreement with previous analyses using srRNA sequences [10, 16, 25, 26]. In addition, we found that Hartmannella grouped with Acanthamoeba, but that the branching order among the three H. vermiformis strains could not be determined. Previous workers grouped Acanthamoeba with the alga Cryptomonas phi (Nu) [10] and found Acanthamoeba branching off from the plant lineage rather than from the human/yeast lineage [23, 26]. Our results for Hartmannella suggest the same is true for this organism (Fig. ЗА), but distances separating the branching points for amoebae, plants, humans and yeast are small. Thus, we are unable to unambiguously determine whether Hartmannella and Acanthamoeba branch with plants, or humans and yeast, or separately. Although the phylogenetic relationships of Acanthamoeba and Hartmannella to higher eukaryotes might seem surprising, it has been pointed out previously that the metamitotic nuclear division of Acanthamoeba, which includes nuclear membrane and nucleolar disintegration and the presence of centrioles during mitosis, closely resembles that of plants and animals [8, 24]. In contrast, nuclear division in the very distantly related amoeba Naegleria is premitotic and the nuclear membrane remains intact during mitosis [3]. Nuclear division in H. vermiformis is mesomitotic, a process more like that in

106 Acanthamoeba; the nuclear membrane disintegrates and the nucleolus disappears, but no centriole-like bodies are observed [3, 24]. The phylogenetic significance of these differences in patterns of mitosis is uncertain, but the differences seem to be consistent with phylogenetic relationships suggested by the srRNA trees.

ACKNOWLEDGMENTS

The authors thank Drs. В.S. Fields and R. Michel for generous gifts of Hartmannella vermiformis strains Atlanta and Koblenz, respectively, Drs. J.H. Gunderson and S.J. Goss for sharing their unpublished srRNA sequence for H. vermiformis, Drs. R. De Wachter and J-M. Neefs for computation and drawing the srRNA secondary structure model for H. vermiformis, and Dr. P. A. Fuerst for helpful advice on analytical approaches and criticism of the manuscript. A travel grant for P.H H. Weekers was provided by the Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO). This research was supported by the Ohio State University and NIH/NEI grant RO1-EY09073 to TJB.

LITERATURE CITED

1 Anzil, A.P., Rao, C, Wrzolek, Μ .Α., Visvesvara, G.S., Sher, J.H. & Kozlowski, P.B. 1991 Amebic meningoencephalitis in a patient with AIDS caused by a newly recognized opportunistic pathogen - Leptomixid amoeba Arch Pathol Lab Med 115 21-25 2 Baverstock, P.R., Illana, S., Christy, P.E., Robinson, P.S. & Johnson, A.M. 1989 srRNA evolution and phylogenetic relationships of the genus Naeglena (Protista Rhizopoda) Mol Biol Evoi 6 243-257 3 Bovee, E.C. 1985 Class Lobosea Carpenter 1861 ρ 158-211 In An Illustrated Guide to the Protozoa, Lee, J J, Hutner, S Η & Bovee, E С (eds ), Society of Protozoologists, Lawrence, Kansas 4 Burg, J.L., Paulethy, СМ., Boothroyd, P. & Gover, J.C. 1989 Direct and sensitive detection of a pathogenic protozoan Toxoplasma gondii by polymerase chain reaction J Clin Microbiol 27 1787-92

107 5 Cabot, EX. & Beckenbach« A.T. 1989 Simultaneous editing of multiple nucleic acid and protein sequences with ESEE Compul Appi Biosci 5 233-234 6 Clarholm, M. 1981 Protozoan grazing on bactena in soil · Impact and importance Mtcrob Ecol 7 343-350 7 Clarholm, M. 198S Possible roles of roots, bactena, protozoa and fungi in suppying nitrogen to plants ρ 355-365 In Fitter. AH & Usher, MB (eds). Ecological interactions in soil Spec Pubi No 4, Br Ecol Soc, Blackwell Scientific Publications Ltd, Oxford 8 Clark, CG. & Cross, G .A.M. 1988 Small-subumt nbosomal RNA sequence from Naeglena gruben supports the polyphyletic origin of amoebas Mol Biol Evol 5 512- 518 9 De Rijk, P., Neefs, J-M-, Van de Peer, Y. & De Wachter, R 1992 Compilation of small nbosomal subunit RNA sequences Nucl Acids Res 20 2075-2089 10 Douglas, S.E., Murphy, CA., Spencer, D.F. & Cray, M.W. 1991 Cryptomonad algae are evolutionary chimaeras of two phylogenetically distinct unicellular eukaryotes Nature 350 148-151 11 Elliot, E.T., Coleman, D.C. & Cole, C.V. 1979 The influence of amoebae on the uptake of nitrogen by plants in gnotobiotic soil ρ 221-229 In Harley, JL & Russell, R S (eds ), The soil-root interface Academic Press, London 12 Fields, B.S., Nerad, ΤΛ., Sawyer, Т.К., King, C.H., Barbaree, J.M., Martin, W.T., Morrill, W.E. & Sanden, G.S. 1990 Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis J Protozoo! 37 581-583 13 Felsenstein, J. 1989 PHYLIP - Phylogeny inference package (Version 3 2) Cladtstics 5 164 166 14 Gunderson, J.H. & Sogin, M.L. 1986 Length variation in eukaryotic rRNAs small- subunit rRNAs from the protists Acanthamoeba castellami and Euglena gracilis Gene 44 63-70 15 Gutell, R.R., Weiser, В., Woese, CR. & Nolier, H.F. 1985 Comparative anatomy of 16S-like nbosomal RNA Prog Nucl Acid Res Mol Biol 32 155-216 16 Johnson, A.M. 1990 Phylogeny and evolution of protozoa Zool Sci 7(Suppl.) 179- 188 17 Johnson, A.M., Fielke, R., Christy, P.E., B.S. Robinson & Baverstock, P.S. 1990 Small-subumt nbosomal RNA evolution in the genus Acanthamoeba J Gen Microbiol 136 1689-1698 18 Lapalme, G., Cedergen, R.J. & Sankoff, D. 1982 An algorithm for the display of nucleic acid secondary structure Nucl Acids Res 10 8351-8356 19 Martinez, A.J. 1985 Free-living amebas natural history, prevention, diagnosis, pathology and treatment of disease CRC Press Ine Boca Raton, Florida.

108 20 Neefs, J-M., Van de Peer, Y„ De Rijk, P„ Gons, A. & De Wachter, R. 1991 Compilation of small nbosomal subunit RNA sequences Nucí Acids Res 19 1987-2015 21 Page, F.C. 1988 A new key to freshwater and soil gymnamoebae Freshwater Biological Association, Ambleside, Cumbria, United Kingdom ρ 14-15 22 Sanger, F. 1981 Determinating of nucleotide sequences in DNA Science 214 1205- 1210 23 Schlegel, M. 1991 Protist evolution and phylogeny as discerned from small-subunit nbosomal RNA sequence comparison Eur J Protistol 27 207-219 24 Singh, B.N. & Hanumaiah, V. 1979 Studies on pathogenic and non-pathogenic amoebae and the bearing of nuclear division and locomotive form and behaviour on the classification of the order Amoebida Ind J Microbiol (Monagr #1) 80 pp 25 Sogin, M.L. 1989 Evolution of eukaryonc microorganisms and their small-subunit nbosomal RNAs Amer Zool 29 487-499 26 Sogin, M.L. 1991 The phylogenetic significance of sequence diversity and length variation in eukaryotic small subunit nbosomal RNA coding regions ρ 175-188 In Warren, L & Koprowski, Η (eds ), New perspectives on evolution Wiley-Liss Ine, New York 27 Turner, D.H., Sugimoto, N., Jaeger, J.A., Longfellow, CE. Freier, S.M. & Kierzek, R. 1987 Improved parameters for prediction of RNA structure Cold Spring Harbor Symp Quant Biol 52 123-133 28 Visvesvara, G.S. & Stehr-Green J.K. 1990 Epidemiology of free-living ameba infections J Protozool 37 25S-33S 29 Zucker, M. & Stiegler, P. 1981 Optimal computer folding of large RNA sequences using thermodynamics and auxilary information Nucí Acids Res 9 133

109

Summary

Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis are free-living amoebae which occur in soil and aquatic habitats but are also known as symbionts and potential pathogens in man and animal. In soil they consume a wide variety of food, especially bacteria, and they play an important role in the mineralization of nitrogen. Representatives of several groups of bacteria including pigment containing, Gram-positive and Gram-negative, and indigenous soil bacteria were used in grazing studies to quantitate amoebal food preferences, growth response and ammonium production (Chapter 2). Food preferences and differences in amoebal growth response and ammonium production were found for Acanthamoeba and Hartmannella. The Enterobacteriaceae, Escherichia coli K12 and Klebsiella aerogenes, proved to be a far better food source than other bacteria like Agrobacterium, Arthrobacter, Bacillus or Pseudomonas, some of which are indigenous to soil. Distinct variations in the production of ammonium were correlated to amoebal growth. Research on the physiology and biochemistry of free-living amoebae has been hampered because of the small number of axenic species available. Axenic cultivation of A. castellana in fermentors (14 1) yielded after 20 days approximately 3 g cells (wet weight) (Chapter 3). After a short lag phase amoebal cell numbers increased to a maximum of 3.5 χ 10' cells per ml. The production of ammonia had a growth inhibitory effect, however, the sudden termination of the exponential growth phase and cell death after 20 days could not be explained by the toxic influence of ammonia only. A simple method for the axenic cultivation of A. castellami and H. vermiformis in chemostats was developed (Chapter 4). Under steady-state conditions minimum generation times of approximately 25 and 16 h and maximum cell densities of 2.5 and 3.2 χ IO6 cells per ml for A. castellami and H. vermiformis were obtained, respectively. Free-living soil amoebae play an active role in the nitrogen mineralization in soil. A number of key enzymes involved in the nitrogen metabolism of A. castellami, A. polyphaga and H. vermiformis were determined (Chapter 5).

Ill Specific enzyme activities revealed that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activities. The highest activities were detected for the transaminase reactions yielding glutamate, and for glutamate dehydrogenase, and a general conclusion was that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes. Lysates of axenically and monoxenically grown A. castellami, A. polyphaga and H. vermiformis were studied by Polyacrylamide isoelectric focusing (Chapter 6). Isoenzyme patterns of acid phosphatase, propionyl esterase, malate dehydrogenase, alcohol dehydrogenase, glucose phosphate isomerase and phosphoglucomutase were compared. Zymograms revealed differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and between axenically grown A. castellana, A. polyphaga and H. vermiformis. Lysates of monoxenic cultures showed a lower intensity and even lack of typical isoenzyme bands as compared to lysates from axenic cultures. Bacteriolytic activities of axenically grown A. castellami, A. polyphaga and H. vermiformis towards various Gram-positive and Gram-negative bacteria were determined (Chapter 7). A spectrophotometrical assay revealed that the specific bacteriolytic activities of both Acanthamoeba species were approximately twice as high as those of the three Hartmannella strains tested. Isoelectric focusing gel-electrophoresis in combination with a newly developed activity assay revealed distinct differences between typical pi points for bacteriolytic activities of Acanthamoeba and Hartmannella. Bacteriolytic activities of Hartmannella were more pronounced and observed in the pi range of 4.0-9.3 while for Acanthamoeba the range was pi 4.5-8.9. Molecular biological techniques were used to sequence the small-subunit ribosomal RNA (srRNA) genes of different Hartmannella strains (Chapter 8). Sequence data were used to reveal differences between the Hartmannella strains, to compare Hartmannella with its closest relative Acanthamoeba and, for phylogenetic placement of these lobosea amoebae among eukaryotes. A secondary structure model for the Hartmannella srRNA was computed in order to compare typical stem and loop structures and variable regions in the phylogenetically closely related Acanthamoeba and Hartmannella.

112 Free-living soil amoebae play an important role in mineralization processes in nature but are only scarcely studied. Simple methods for the mass cultivation of axenic cells were established and cells were used to study amoebal-bacterial interactions, amoebal enzymes and phylogenetic relationships among amoebae. The research described in this thesis can be a step foreward towards more extensive physiological, biochemical and molecular biological investigations of these free-living amoebae.

113 Samenvatting

Acanthamoeba castellami, Acanthamoeba polyphaga en Hartmannella vermiformis zijn vrij-levende naaktamoeben die voorkomen in de bodem en aquatische habitats, maar ook als symbionten en zelfs als potentieel pathogenen bij mens en dier. In de bodem begrazen ze de microbiele populatie waar ze zich vooral voeden met bacteriën. Door het begrazen van deze micro-organismen spelen ze een belangrijke rol in de mineralisatie van stikstof in de bodem. Verschillende soorten bacteriën waaronder pigment bevattende, Gram-positieve en negatieve, en typische bodem-bacteriên werden in begrazings-studies gebruikt om de voedselvoorkeur, de groei respons en de ammonium productie van deze amoeben te bepalen (Hoofdstuk 2). Tussen beide genera, Acanthamoeba en Hartmannella, werden verschillen aangetoond. In groei-opbrengst experimenten bleken de niet-pigment bevattende Enterobactenaceae zoals Escherichia coli K12 en Klebsiella aerogenes een veel betere voedingsbron te zijn dan andere bacteriën zoals Agrobactenum, Arthrobacter, Bacillus of Pseudomonas. Variatie in de geproduceerde hoeveelheid ammonium was het gevolg van verschillen in voedselvoorkeur bij de begrazing. Fysiologisch en biochemisch onderzoek aan vrij-levende amoeben werd bemoeilijkt doordat maar weinig soorten in axene cultuur zijn gebracht. Het axeen kweken van A. castellami in fermentoren (14 1) leverde binnen 20 dagen ongeveer 3 g natgewicht aan cellen (Hoofdstuk 3). Na een aanloop fase nam het aantal cellen toe tot ongeveer 3.5 χ 10' per ml. De vorming van ammonia had een groeiremmend effect maar was geen voldoende verklaring voor het plotselinge afsterven van de cellen na 20 dagen. Een eenvoudige en praktische methode werd ontwikkeld voor het kweken van A. castellami en H. vermiformis in een chemostaat (Hoofdstuk 4). Onder evenwichtsomstandigheden werd een minimum generatietijd bereikt van respectievelijk 25 en 16 uur, en een maximum celdichtheid van respectievelijk 2.5 en 3.2 χ 106 cellen per ml voor A. castellami en H. vermiformis. De vrij-levende bodemamoeben gebruiken een verscheidenheid aan voedsel en spelen een belangrijke rol in de mineralisatie van stikstof in de bodem.

114 Sleutelenzymen van het stikstof metabolisme van A. castellana, A. polyphaga en H. vermiformis werden aangetoond (Hoofsdtuk 5). Uit de specifieke enzym activiteit bleek dat alle amoeben uraat oxidase, glutamine synthetase, NADP- afhankelijk glutamaat dehydrogenase, glutamaat oxaloacetaat transaminase en glutamaat pyruvaat transaminase activiteit bevatten. De hoogste activiteiten werden aangetroffen voor de transaminase reacties die glutamaat opleverden en voor het glutamaat dehydrogenase. De conclusie was dat de route voor stikstof assimilatie van deze vrij-levende amoeben gelijk is aan die van andere eukaryoten. Axeen en monoxeen gekweekte A. castellana, Α. polyphaga en H. vermiformis werden gelyseerd en met behulp van Polyacrylamide isoelectrische focusing onderzocht (Hoofdstuk 6). Isoenzym patronen voor zure fosfatase, propionyl esterase, malaat dehydrogenase, alcohol dehydrogenase, glucose fosfaat isomerase and fosfoglucomutase werden vergeleken waarbij de zymogrammen verschillen in typische isoenzym patronen tussen axeen en monoxeen gekweekte amoeben te zien gaven. Bij lysaten van monoxeen gekweekte amoeben werd in vergelijking met lysaten van axeen gekweekte amoeben een lagere intensiteit en zelfs het ontbreken van typische isoenzym banden aangetoond. Het spectrum van de axeen gekweekte A. castellana, Α. polyphaga en H. vermiformis voor de bacteriolyse van verschillende Gram-positieve en Gram- negatieve bacteriën werd bepaald (Hoofdstuk 7). Met behulp van een spectrofotometrische methode werden verschillen in specifieke bacteriolytische activiteiten tussen Acanthamoeba en Hartmannella aangetoond. Met behulp van een isoelectrische focusing techniek in combinatie met een nieuw ontwikkelde activiteitstest werden verschillen tussen de bacteriolytische activiteiten van de amoeben aangetoond. De typische bandenpatronen met bacteriolytische activiteiten waren bij Hartmannella te zien als strakke bandjes in het pi gebied 4.0-9.3 terwijl deze bij Acanthamoeba in het pi gebied 4.5-8.9 lagen. Moleculair biologische technieken werden toegepast om de kleine sub- eenheid van het ribosomaal RNA (srRNA) gen van verschillende Hartmannella stammen te sequencen (Hoofdstuk 8). Deze gegevens werden gebruikt om verschillen tussen de Hartmannella stammen onderling en verschillen tussen Hartmannella en het meest verwante organisme Acanthamoeba aan te tonen, en om beide Iobose amoeben fylogenetisch binnen de eukaryote organismen te

115 plaatsen. De secundaire structuur van het srRNA werd bepaald om op basis van typische stem/loop structuren en variable regio's de fylogenetisch nauw verwantte Acanthamoeba en Hartmannella te kunnen vergelijken.

Vrij-levende naaktamoeben spelen een belangrijke rol in de mineralisatie- processen in de natuur, maar zijn tot dusver nog relatief weinig bestudeerd. In dit proefschrift is beschreven hoe met behulp van eenvoudige en doeltreffende kweekmethoden een voldoende hoeveelheid axene cellen kunnen worden verkregen. Met deze cellen zijn zowel begrazings-, enzym-, als fylogenetische studies uitgevoerd. Hiermee is een aanzet gegeven tot verder fysiologisch, biochemisch en moleculair biologisch onderzoek aan deze vrij-levende amoeben.

116 Dankwoord

Mijn proefschrift is dan eindelijk af. Het is er duidelijk anders uit komen te zien dan bij de aanvang van mijn promotie in October 1987 voorzien kon worden. Onderzoek aan vrij-levende bodemamoeben vond op dat moment in Nederland niet plaats. Mijn promotor, Fried Vogels, hield me dan ook altijd voor dat met zo een "open" promotie-onderwerp de mogelijkheden voor het oprapen lagen. Ondanks dit vermeende gemak bleek het onderzoek aan deze vrij-levende bodemamoeben toch niet zo gemakkelijk te zijn als aanvankelijk gedacht. Het werken in team verband binnen "onze protozoën-groep", maar ook de samenwerking met laboratoria in het buitenland hebben een wezenlijke bijdrage geleverd aan het tot stand komen van dit proefschrift. Allereerst wil ik "mijn" studenten, Bart Lomans, Paul Bodelier, John Wijen, Hans Idink, Ruud Nabben en Andreas Engelberts, bedanken. Niet alleen voor het mede uitvoeren van de experimenten maar vooral ook voor de bijzonder prettige manier waarop we hebben kunnen samenwerken. Ook jullie moesten vaak echt "tot de bodem gaan". Mijn promotor, Fried Vogels, wil ik bedanken voor de vrijheid die ik kreeg bij het uitvoeren van dit onderzoek en voor de vele waardevolle discussies met betrekking tot dit ook voor hem nieuwe onderzoeksgebied. Verder dank ik alle (ex)-collega's, (ex)-studenten en (ex)-stagaires op de afdeling Microbiologie voor de goede sfeer en prettige samenwerking. Met name wil ik speciaal Chris van der Drift bedanken voor de ondersteuning bij "die vier extra maanden". De Programma Commissie Basiskennis Bodemonderzoek (PCBB) bedank ik voor de financiële ondersteuning tijdens de eerste vier jaren van het promotie­ onderzoek, waarbij ik tevens de twee thema-coördinatoren van de onderzoekslijn C-8 wil bedanken voor hun kritische adviezen. Met name wil ik hierbij Riks Laanbroek bedanken voor zijn waardevolle suggesties en ondersteuning "op het juiste moment". Johan De Jonckheere van het Instituut voor Hygiene en Epidemiologie in Brussel (België) wil ik bedanken voor de vruchtbare samenwerking en zijn "goede invloed" op mijn promotie-onderzoek. Niet alleen het amoeben-congres dat je in 1989 organiseerde maar ook de verschillende keren dat ik op jouw

117 laboratorium in Brussel heb mogen werken waren van groot belang voor mijn promotie-onderzoek. Daarnaast waren ook de "werkbesprekingen 's avonds" bijzonder aangenaam. Thomas Byers, I would like to thank you for the opportunity to work in your laboratory at the Department of Molecular Genetics of The Ohio State University (Columbus, Ohio, USA) and for the fruitful collaboration. I also would like to thank you and your wife Sandy for the hospitality during the time that I stayed as a guest in your house. I also thank all the people from "the second floor" of the Biological Sciences building of OSU, and especially Becky who had to spent a lot of time on teaching me molecular biology and phylogenetics. De afdeling grafische vormgeving met name Jan Peters, John Slippens en Hanske van Westreenen, en de afdeling fotografie met name Gerard Dekkers en H. Spruyt wil ik bedanken voor de goede en vaak zeer snelle verwerking van mijn illustraties. Al mijn vrienden, en vooral "de Luxemburg club", die ik noodgedwongen gedurende de slotfase van mijn promotie-onderzoek heb moeten verwaarlozen. Ik hoop dat jullie konden begrijpen dat mijn afwezigheid niet uit gemakzucht of desinteresse maar uit noodzaak voortkwam. Mijn familie en met name mijn ouders dank ik voor hun aanmoediging om maar vooral verder te studeren en voor hun steeds aanwezige steun en vertrouwen gedurende deze promotie-periode. And last but not least Becky thanks for all your support. ">iW

118 Curriculum vitae

De schrijver van dit proefschrift werd op 2 juni 1959 geboren te Weert. In 1976 werd het diploma HAVO aan het Bisschoppelijk College te Weert behaald. In 1982 werd de tweedegraads onderwijsbevoegdheid voor biologie en aardrijkskunde aan het Mollerinstituut te Tilburg behaald. In datzelfde jaar zette hij zijn studie biologie voort aan de Katholieke Universiteit Nijmegen. In juni 1987 werd het doctoraal examen afgelegd met als hoofdvak Microbiologie (Prof. Dr. Ir. G.D. Vogels en Dr. L.G.M. Gorris); hierbij werden methanogene cofactoren getest om als parameters te dienen voor het monitoren van UASB reactoren. Tijdens het eerste bijvak Exobiologie (Prof. Dr. A.W. Schwartz en Dr. CG. Bakker) werd onderzoek gedaan naar de mogelijke prebiotische synthese routes en de uitgangsproducten voor de synthese van 2'-nor-2'- deoxyguanosine. Tijdens het tweede bijvak Dierfysiologie (Prof. Dr. S.E. Wendelaar-Bonga en Dr. P. van der Meijden) werd, bij Organon International te Oss, de invloed van inducers en de rol van het cytochroom P-450 bij de 11a- hydroxylering van epoxyprogesteron in het Πα-hydroxylase systeem van Aspergillus ochraceus bestudeerd. Van October 1987 tot October 1991 was hij als wetenschappelijk medewerker werkzaam op het laboratorium voor Microbiologie van de Katholieke Universiteit Nijmegen in het kader van een door de Programma Commissie Basiskennis Bodemonderzoek (PCBB) gefinancierd onderzoek. Daaropvolgend was hij van October 1991 tot februari 1992 werkzaam als wetenschappelijk medewerker op hetzelfde laboratorium in het kader van een door dat laboratorium gefinancierd vervolgonderzoek. Gedurende korte periodes in september en november 1991 was hij werkzaam op het Instituut voor Hygiene en Epidemiologie (Brussel, België), en gedurende februari tot en met april en in augustus en september 1992 was hij werkzaam op het Department of Molecular Genetics van de Ohio State University (Columbus, Ohio, USA). Vanaf 1 mei 1993 is hij als wetenschappelijk medewerker in tijdelijke dienst verbonden aan de Vakgroep Microbiologie en Evolutiebiologie van de Katholieke Universiteit Nijmegen.

119

Publications

Weekers, P.H.H. & Van der Drift, С. 1993. Nitrogen metabolizing enzyme activities in the free-living soil amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis. J. Euk. Microb. 40:251-254. Weekers, P.H.H., Bodelier, P.L.E., Wijen, J.P.H. & Vogels, G.D. 1993. Effects of grazing by the free-living soil amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella -vermiformis on various bacteria (Submitted for publication). Weekers, P.H.H., Engelberts, A.M.W. & Vogels, G.D. 1993. Bacteriolytic activities of the free-living soil amoebae, Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis (Submitted for publication). Weekers, P.H.H., Gast, R.J. & Byers, T.J. 1993. Small-subunit ribosomal RNA of three Hartmannella vermiformis strains. Sequences, secondary structure and phylogenetic implications (Submitted for publication). Weekers, P.H.H., Wijen, J.P.H., Lomans, B.P. & Vogels, G.D. 1993. Mass cultivation of the free-living soil amoeba, Acanthamoeba castellani! in a laboratory fermentor (Submitted for publication). Weekers, P.H.H. & De Jonckheere, J.F. 1993. Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella (Submitted for publication). Weekers, P.H.H. & Vogels, G.D. 1993. Axenic cultivation of the free-living soil amoebae, Acanthamoeba castellami and Hartmannella vermiformis in a chemostat (Submitted for publication).

Abstracts

Weekers, P.H.H. Weekers & Vogels, G.D. 1989. Isolation, cultivation and nutritional requirements of soil amoebae. In: Abstracts of the 2n

121 Weekers, P.H.H., Lomans, B.P. & Vogels, G.D. 1990. Utilisation of substrate, oxygen consumption and ammonia production by soil amoebae. In: Abstracts of the 3* Symposium of The Netherlands Integrated Program for Soil Research (PCBB). Lunteren, November 26, 1990 - November 28, 1990. (Abstract C8-4/8938). Weekers, P.H.H., Bodelier, P.L.E., Wijcn, J.P.H. & Vogels, G.D. 1992. Differences in growth rates and ammonia excretion of soil amoebae feeding on various food bacteria. J. Protozool. 39:17A. 10. Wissenschaftlichen Tagung der Deutschen Gesellschaft für Protozoologie e.V., 20.-23. März 1991 in Gießen, Germany. (Abstract 97). Weekers, P.H.H., Wijen, J.P.H. & Vogels, G.D. 1991. Differences in the growth capacity, and ammonia production of the soil amoebae; Acanthamoeba castellami, Acanthamoeba polyphaga and Hartmannella vermiformis, grazing on various food bacteria. In: Abstract of the 4* Symposium of The Netherlands Integrated Program for Soil Research (PCBB). Lunteren, November 18, 1991 - November 20, 1991. (Abstract C8- 4/8938). Weekers, P.H.H., Gast, R.J. & Byers, T.J. 1993. Small-subunit ribosomal RNA sequences of three strains Hartmannella vermiformis. J. Euk. Microbiol. 40:47A. Abstract of the VI. International Conference on the Biology and Pathogenicity of Free-Living Amoebae, August 2-7, 1992. Virginia Commenwealth University, Richmond, Virginia, USA. (Abstract 282).

122