Evidence that Thraustochytrium is unable to synthesize lysine

4 70

Mycol. Res. 92 (4): 470-476 (1989) Printed ill Great Brituin --

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Evidence that Thraustochytrium is unable to synthesize lysine

F . M. P A T O N A N D D. H . JENNINGS Depnrtment of Botuny, University of Liverpool, P.O. Box 147, Liverpool L.69 3BX

Evidence that Thraustochytrium is unable to synthesize lysine. Mycological Research 92 (4): 470-476 (1989). When sporangia of Thraustochytrium aureum were grown in the presence of ~-[4-'~C]as~artic acid and [z-14C]acetatevirtually no radioactivity was found in the lysine either free or bound in protein. No activity of saccharopine dehydrogenase (NAD+,lysine requiring) could be detected in extracts of the sporangia. These results indicate that neither the aminoadipic acid nor the diaminopimelic acid pathway are present in this organism. Attempts to confirm these findings by other growth studies using T. aureum and T. roseum were inconclusive. The results confirm previous investigations which appear to point to the conclusion that Thraustochytrium and related organisms are not fungi. Key words: Thraustochytrium, Lysine biosynthesis, Evolution. The family Thraustochytriaceae was described by Sparrow (1943) and amended (Sparrow, 1960) to include the genera Thraustochytrium Sparrow (1936). and ]aponochytrium Kobayashi & Ookubo (1953) and later Schizochytrium Goldstein & Belsky (1964). The Thraustochytriaceae were originally placed in the Saprolegniales (Oomycetes) mainly on morphological criteria, i.e. zoospore formation, behaviour and flagellation. They possess biflagellate zoospores, heterokont in morphology and also possibly in flagellar length (Darley & Fuller, 1970). Zoospores are always delimited within the zoosporangia. The Thraustochytriaceae are separated, however, from other members of the Saprolegniales by their vegetative non-productive thallus which is monocentric, polar and eucarpic. Further evidence indicating dissimilarities between the Thraustochytriaceae and the Saprolegniales came from the work on the composition and mode of formation of the cell covering (Darley, Porter & Fuller, 1973). Unlike the Saprolegniales, the principal sugar from the hydrolysis of the outer covering of Schizochytrium aggregatum Goldstein & Belsky and Thraustochytrium spp. is galactose and there is also a large protein fraction. Chamberlain (1980) found no neutral or phospho-lipids and the primary sugars were galactose and xylose (2: 1)with traces of glucose. The presence of sulphated polysaccharide and calcium was indicated but no chitin or cellulose could be detected by the usual biochemical tests. The sulphated polysaccharide, presumably galactan or xylogalactan, shows more algal than fungal affinity (Chamberlain, 1980) as it is a common feature of algal cell walls (Percival & McDowell, 1967; Siegel & Siegel, 1973). Equally important is the fact that the thraustochytrid outer covering, unlike any fungal cell wall, has been found to be multilamellate consisting

of layers of Golgi-derived scales (Darley et al., 1973; Perkins, 1976). The Thraustochytriaceae was later given ordinal status (Thraustochytriales) by Sparrow (1973), was redefined by Alderman et al. (1974) and comprises the genera Thrawtochytrium, ]aponochytrium, Schizochytrium, Althornia, Aplanochytrium and Ulkenia as well as the new thraustochytrid Corallochytrium limacisporium gen. et sp. nov. (Raghu-kumar, 1987). There is no doubt that Thraustochytriales and the seemingly related order Labyrinthulales present considerable difficulties with regard to their taxonomy even at higher levels of classification (Bahnweg & Jackle, 1986). Hori, Lin & Osawa (1985) used the available 5S ribosomal RNA data to construct a phylogenetic tree, and found the thraustochytrids to be a very old group which apparently evolved before the fungi, protozoa, brown algae and green plants. This is in keeping with the findings of Huysmans et al. (1983) who constructed a similarity phenogram from sequences of 5 s ribosomal RNA including those from Ulkenia visurgensis (Ulken) Gaertner and Schizochytrium aggregaturn (MacKay & Doolittle, 1982). The dissimilarity between the thraustochytrids and other protists was surprisingly high. The majority of studies to date concerning the thraustochytrids, with respect to their classification and taxonomic affinities, have been concerned with morphology and ultrastructure. Few studies have been concerned with the biochemical and physiological characteristics of these organisms. It is particularly notable that there has been, as yet, no attempt to examine the pathway of lysine biosynthesis, information about which has been used to good effect by Le John (1971) to suggest phylogenetic relationships within the Fungi. It is known that there are two pathways of lysine

F. M. Paton and D. H. Jennings biosynthesis in nature, one involving a-arninoadipic acid (AAA) and the other involving a,~-diaminopimelic acid (DAP). The DAP pathway has been found to operate in pseudomonads, eubacteria, actinomycetes (Vogel, 1959a), cyanobacteria (Vogel, 19596), pennate marine diatoms (Brown & Cooksey, 1984), bryophytes and tracheophytes (Vogel, 1959c) and in those phycomycetes possessing biflagellate or posteriorly uniflagellate zoospores (Vogel, 1964). On the other hand, within the Basidiomycotina and Ascomycotina and those phycomycetes possessing nonflagellate spores or anteriorly uniflagellate zoospores, lysine is synthesized via the AAA pathway (Vogel, 1964). Since the evidence to date (Bahnweg, 1979) indicated that species of Thraustochytrium do not require lysine for growth, we decided to investigate the pathway by which the amino acid is synthesized. While elucidHtion of the lysine biosynthetic pathway in Thrawtochytrium sp. will not necessarily resolve the taxonomic affinities, the presence of the AAA would suggest an evolutionary relationship of the genus with the Euglenophyta and higher fungi. The presence of the DAP pathway would argue against such a relationship. In the event, the evidence which has come forward suggests that contrary to what had been believed, Thrawtochytrium does not seem to be able to synthesize lysine.

MATERIALS A N D METHODS Organisms Thraustochytrium aureum Goldstein and T. roseum Goldstein were received from Dr S. T. Moss, School of Biological Sciences, Portsmouth Polytechnic.

Media GTYSW medium. Glucose, 5 g; tryptone (Oxoid), 2 g; yeast extract (Oxoid), 1g; sea water 1 1; agar (Oxoid) (when present) 10 g. GGVSW medium. Glucose, 5 g; glutamic acid, 5 g; vitamin B,, 0.01 mg; vitamin B,, 0.01 mg; biotin, 0.01 mg; vitamin B,,, 0.05 mg; sea water, 11 adjusted to pH 7-0 with sodium hydroxide. Glucose/amino acid medium. Glucose, 5 g; L-alanine, 0.052 g; L-arginine(monohydrochloride), 0.062 g; DL-aspartic acid, 0.136 g; diaminopimelic acid, 0.006 g; L-glutamic acid, 0.346 g; L-isoleucine, 0.076 g ; L-leucine, 0.138 g ; L-lysine (monohydrochloride), 0.158 g; L-proline,0.170 g; L-threonine, 0.046 g ; sea water, 1 1. Artificial sea water. NaCl, 24 g; MgSO,. 7H20, 12 g ; CaC1,. 2H,O, 1 g; KCI, 0.75 g ; NaNO,, 0.04 g; K,HPO,, 0.001 g; tris, 1 g; Na,EDTA, 12 mg; ZnS0,. 7H,O, 2 mg; NaMoO,. 2H20, 1mg; FeCl, .6H20,'0.5 mg; MnCl, .4H20, 0.2 mg; CoC1,. 6H20, 2 pg; CuS0,. 5H,O, 2 pg; distilled water, I 1, plus the following made up in a separate solution which was filter sterilized before being added to the autoclaved solution of the above constituents: NaHCO,, 0.1 g; thiamine hydrochloride, 300 pg; p-amino-benzoate, 20 pg; calcium pantothenate, 10 pg; cyanocobalamin, 4 pg; distilled water, 1 I.

Cultures Stock cultures. These were maintained on GTYSW agar (10 ml) slopes in McCartney bottles in the dark at 20 OC. When the bottles were inoculated 10ml of sterile sea water were introduced at the same time. The bottles were inverted weekly to aid dispersal of zoospores. Liquid cultures. 2 ml of sea water containing zoospores from stock cultures were transferred to 200 ml GTYSW medium in 500 ml Erlenmeyer flasks which were then incubated on an orbital shaker at 100 rev. min-l in the dark at 20 O. The organisms were subcultured weekly by transferring 10 ml aliquots of the culture suspension into 200 ml of fresh medium. When inocula were required for experimental purposes, 3-d-old cultures were used.

Erlenmeyer flasks (100 ml) containing 20 ml of either GTYSW or GGVSW medium were inoculated with 5 ml of a liquid culture. At the same time 555 kBq of the labelled compound in sterile distilled water was added, then the flasks were sealed with subaseal rubber bungs pierced by h e aluminium wire to which the attached 5 ml glass vessels suspended in the neck of the culture flasks. The vessels contained 3 ml 30% (w/v) potassium hydroxide to absorb radioactive carbon dioxide. The flasks were then incubated in a shaking water bath for 3 d in the dark at 20'.

Harvesting the organism Sporangia were separated from media by vacuum filtration under reduced pressure through 10 pm nylon mesh. The sporangia were washed with 25 ml of distilled water at 4 O scraped off the mesh with a spatula, placed in pre-weighed aluminium foil boats and dried to constant weight at 70 O.

Protein hydrolysis and amino acid analysis The method used was adapted from that of Roberts et al. (1955). A known dry weight of between 0.15 and 0.20 g Thrawtochytrium was placed in a hydrolysis tube with 4-5 ml 6 N HCL (double distilled) which was frozen and thawed under high vacuum three times to remove any oxygen, then sealed under vacuum and placed in an oven at 110 O for 20 h. The hydrolysate was evaporated to dryness on a rotary evaporator in a 50 ml round-bottomed flask and washed twice with distilled water. Finally, the samples were resuspended in 30 ml citrate buffer, pH 2.2. Amino acid analysis was carried out using a JEOL JAC 6AH amino acid analyser.

Decafionization Analysis of distribution of 14Camongst amino acids required that extracts be decationized. This was achieved by passing 5 ml aliquots through 7 ml columns of Dowex 50W 20+400 mesh strongly acidic cation exchange resin. The resin was charged with 50 ml 5 % (w/v) HC1 and rinsed with

Thrausfochytriurn and lysine synthesis 2 x 100 ml deionized water. The columns were subsequently eluted with 40 ml 8 N ammonia followed by a 50 ml deionized water wash and this eluate was collected, evaporated to dryness as described above and made up in 100 yl ethanol for thin layer chromatography. Recovery of all amino acids was 100f 5 % other than aspartic acid, glutamic acid and methionine whose recovery was 85 f 5 %. However, as this latter value was found to be consistent over three separate estimations, a correction factor was employed in all calculations.

RESULTS

When Thraustochytrium aureurn was grown on GTYSW medium at 20' in the dark, maximum dry weight was achieved after 4 d. O n GGVW medium, maximum dry weight was achieved after 5 d and the amount of dry matter produced was only 30% of the maximum achieved in GTYSW. Successive subculturing from GGVSW medium into fresh GGVSW medium resulted in a cessation of growth. It could be shown that sporangia remained viable by subculturing from such media in which no growth had taken place to GTYSW medium when growth occurred after a lag of almost 24 h. Thin layer chromatography and autoradiography From these observations, it was decided that, when it was Aliquots of the extracts in ethanol were applied to one comer necessary to produce sporangia by growth in GGVSW of 20 x 20 cm plastic-backed plates covered with a layer medium, sporangia from GTYSW medium would be used as 250 pm thick of cellulose MN 300 (Machery-Nagel Polygram the inoculum to ensure reasonable growth and hence dry Cel 300) which were run in two dimensions-1st 2-proponal: matter. ethylmethyl ketone (butanone): 1 N HC1 (60: 15 :25); 2nd Table 1 shows the total amount of amino acids after 2-methyl-2-butanol: ethylmethyl ketone: propanone: hydrolysis of sporangia of T. aureurn after growth on GTYSW methanol: water: ammonia (0.88) (50: 20: 10: 5 :15 : 5). The or GGVSW media and of T. roseurn after growth on GTYSW. solvent was evaporated from the plate at room temperature Addition of a known weight of lysozyme to the dried after each run. When required, the amino acids were identified sporangia before hydrolysis and correcting for the amino acid by spraying with ninhydrin-cadmium acetate reagent (0.5 g content of the added lysozyme showed that the procedures cadmium acetate in 50 ml H,O to which 20 ml glacial acetic used result in efficient hydrolysis of protein. acid had been added). Acetone was added to 500 ml and when The values for the content of the various amino acids required, ninhydrin was dissolved in portions of this to a final represent not only that coming from the hydrolysis of protein concentration of 0.2 % (w/v). The plates were then examined but that due to amino acids free in the cytoplasm. A previous by autoradiography. study (Wethered & Jennings, 1985) showed that the majority of soluble amino acids in T. aureurn are at a content of less than 15 ymol g-l dry weight with the exception of proline Determination of radioactivity (116 pmol g-l dry weight) glutamic acid (29), aspartic acid Radioactive material was scraped off the plates into 10 ml (20) and alanine (16). A known weight of T. aureurn was Beckrnan MPE scintillation cocktail and the radioactivity homogenized in a mortar with a pestle with 5% (w/v) cold determined. trichloracetic acid and rinsed with ethanol followed by ether. The washings were combined and evaporated to dryness Extraction and assay of saccharopine dehydrogenase under reduced pressure and resuspended in citrate buffer, W A D + lysine requiring) (EC 7 . 5 . 7 . 7) pH 2.2. The analysis of amino acids in this extract showed that only the following amino acids had values greater than Three-d-old cultures of T. aureurn growing in GGVSW were 20 vmol g-' dry weight - proline (ll8),'glutamic acid (71), homogenized in an X-press with 0.1 ~-tris-HClbuffer pH 7.4 glycine (38), lysine (27), aspartic acid (26) and alanine (21). It at 4'. Cell debris was removed by centrifugation at 1400 g for is clear from these values that of the pool of free amino acids, 20 min. The supernatant was dialyzed at 4' for 12 h against only proline and glutamic acid make any significant contribu40 volumes of buffer consisting of 5 m~-KH,PO,, 1mM tion to the total content of amino acid after hydrolysis. 2-mercapto-ethanol, 0.1 m~-EDTA,pH 7.0, then centrifuged When T. aureurn was grown in GTYSW medium in the as before. The final supernatant was diluted to 13 mg ml-' acid, label was only detectable in presence of ~-[4-'~C]as~artic protein with pH 7.0 buffer. Saccharopine dehydrogenase aspartic acid, glutamic acid and alanine with trace amounts of activity was assayed at 25' by measuring the lysine, radioactivity in threonine (Table 2). No label was detectable in a-ketoglutarate-dependent oxidation of NADH spectrophotolysine. The presence of label in aspartic acid, glutamic acid and metrically. Incubation mixture contained 100 p~ L-lysine; alanine is similar to what has been found for urediniospores of 20 PM a-ketoglutarate; 250 IJM potassium phosphate buffer, rust fungi (Staples, Birchfield & Baker, 1961). The absence of pH 7.0; 0.25 p ~ - N A D H ;sufficient enzyme and water to label in threonine and lysine might be due to feedback adjust the final volume to 2 ml. Readings were taken every inhibition owing to the presence of these amino acids supplied 30 s for 3 min at 340 nm. A unit of enzyme is the amount from tryptone in the growth medium in a manner similar to which catalyses the oxidation of NADH at a rate of 0.01 O D that found for a variety of green plant tissues (Miflin et al., unit min-' at 25'. 1979). Table 2 shows that when T.aureurn was grown in the Labelled compounds presence of L-[4-'*C]aspartic acid in GGVSW medium, in L-[4-14C]asparticacid and [2-14C]sodiumacetate were obtained which the source of nitrogen was solely glutamic acid, label was found in aspartic acid, threonine, glutamic acid, alanine from Amersham International plc.

F. M. Paton and D. H. Jennings

4 73

Table 1. Free and protein amino acid content (urn01 g-' dry weight fs.E.)of the sporangia after hydrolysis of ntrausto~h~trium aureum after growth on GTYSW and GGVSW and T. roseum after growth on GTYSW

T. aureum GTYSW Amino acid

Content

T. roseum GTYSW

GGVSW Ratio*

Content

Ratio*

Content

Ratio*

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine

* Lysine content taken as unity.

Table 2. Incorporation of 14C from ~-[4-l~C]aspartic acid and [2-14C]acetateinto free and protein amino acids of sporangia of Thraustochytrium aureum after growth on GTYSW or GGVSW. Radioactivity is given as MBq mol-' Sample

Amino acid

1 Specific activity

Aspartic acid Glutamic acid Alanine

0.56 0.24 0.33

Aspartic acid Threonine Glutamic acid Alanine Proline

13.7 2.60 1.08 1.92 1.34

Aspartic acid Threonine Glutamic acid Lysine

6 67 13.9 14.8

2 Specific activity

3 Specific activity

L-[4-14C]asparticacid; GTYSW 30.7 24.2 -

L-14-'"]aspartic

acid; GGVSW

[2-14C]acetate

ND, not detectable;

ND

44.00 20.3 9-14 1.66

50.71 16.5 7.99 2.25

-, no data obtained.

and proline in the percentage ratio 100, 19, 7, 14, 10. Thus it appears likely that feedback inhibition of threonine synthesis has been occurring during growth in GTYSW medium. Further, since no label was found in lysine it seems unlikely that the diaminopimelic acid pathway was operating. When T.aureum was grown in the presence of [2-14C]acetic acid in GGVSW medium, label was present in aspartic acid,

threonine and glutamic acid with trace amounts discemable in lysine in two out of three replicate samples (Table 2). The percentage ratios were respectively 100, 34, 20 and 4. The extremely small amount of label in lysine suggests that if the aminoadipic acid pathway is operating in T. aureum it is not doing so to any significant extent. An attempt to demonstrate activity of the enzyme

Thrausfochytriurn and lysine synthesis Table 3. Extent of growth of sporangia of Thrawtochytrium aureum and T. roseum in various media and their effect on viability of the sporangia T. roseurn

7. aureum

Medium

Growth

GTYSW GGVSW 1

+ +

SMLD SMD SML SM GTSW GVSW

2 3

Viability

Growth

Viability

-

-

-

+ +

+,

Measurable growth; -, no growth or sporangia not viable; * sporangia remain viable.

GTYSW: glucose, tryptone, yeast extract, sea water. GGVSW: glucose, glutamic acid, vitamin supplement, sea water. GTSW: glucose, tryptone, sea water. GVSW: glucose, vitamin supplement, sea water. 1, 2 & 3 represent successive subculturings from GGVSW into fresh GGVSW. S: filtered sea water; M: defined medium; L: lysine present; D: diaminopimelic acid present. For other details about media see text.

saccharopine dehydrogenase, which catalyses the final step of the synthesis of lysine via the aminoadipic acid pathway and involving the oxidative cleavage of saccharopine, proved negative. Those organisms which have no lysine biosynthesis are found in the Protozoa and Metazoa (Brown & Cooksey, 1984). Kidder & Dewey (1945) showed that amongst other amino acids, the single omission of lysine from otherwise adequate growth medium prevents the growth of Tetrahymena geleii Furgason, while Guttmann (1967) showed that of 16 kinds of flagellated protozoan trypanosomatids (which did not contain endosymbionts capable of synthesizing lysine within their bodies) all required lysine for growth; nine would grow in media containing diaminopimelic acid, but not amiadipic acid, instead of lysine. Onodera & Kandatsu (1974) and Ondera, Shinjo & Kandatsu (1974) subsequently showed that rumen ciliates were able to synthesize lysine from DAP added to the growth medium and from DAP contained in rumen bacterial cells but were unable to synthesize lysine from [U-14C]acetate,L-[U-14C]aspartateof [6-14C]aminoadipicacid. Taking into account the above information and information on the amino acid composition of tryptone and yeast extract (Oxoid), a glucose/amino acids medium was devised as detailed in the Methods section. Five amino acids found to be essential for the growth of T. geleii and present in tryptone and yeast extract were omitted from the defined medium methionine, phenylalanine tryptophan, histidine and valine as the former three were present in trace amounts in L-leucine while histidine and valine are known not to stimulate growth

of Thrausfochyfriurnaureurn (Bahnweg, 1979). The extent of growth in this medium in the presence or absence of diaminopimelic acid and lysine was investigated. Table 3 shows the result obtained. Growth of both T. aureurn and T. roseurn was only observed in GTYSW and the first subculture into GGVSW. In all other media investigated, there was no measurable growth, though, apart from the third subculture into fresh GGVSW, sporangia remained viable in all media. Addition of lysine at 0 1 5 8 g I-' (the concentration at which it is present in GTYSW) to non-growing cultures did not stimulate growth in any instance. It can be seen also that there did not appear to be in filtered sea water a factor inhibitory to growth (Borut & Johnson, 1962; Kird, 1980) but whose effect was masked by the high carbon and nitrogen content in GTYSW. Artificial sea water was substituted for filtered sea water without any stimulation of growth.

DISCUSSION All organisms capable of synthesizing lysine studied to date have been shown to possess either the a-aminoadipic acid (AAA) or the a,€-diaminopimelic acid (DAP) pathway of lysine biosynthesis. The presence of these pathways has been demonstrated either by extracting and assaying the enzymes involved, or by the use of the relevant 14Clabelled compounds such that lysine is or is not labelled according to the pathway of synthesis. In this study, the presence of ~ - [ 4 - ~ ~ C ] a s ~ a r t i c acid (a specific tracer for the DAP pathway) or [2-14C]acetate (a specific tracer for the AAA pathway) in the growth medium of Thrawfochyfrium aureurn did not lead to any significant label in lysine within the organism. Although there was a very small amount of label in lysine after growth in the presence of [2-14C]acetate, there was no evidence in extracts from sporangia of saccharopine dehydrogenase (NAD+, lysine forming) which enzyme is necessary for the final step of the AAA pathway in higher fungi (Saunders & Broquist, 1966). Consequently it appears that T. aureurn, unlike those fungi studied to date, is unable to synthesize lysine via either the DAP or the AAA pathways. Before commenting on this conclusion, some further amplification of the results is necessary. Vogel (1964) found that incorporation of label into protein asparate or threonine tended to approximate to the same relative specific activity on a molar basis regardless of the tracer used or the mode of lysine synthesis. This could only apply if labelled aspartate was not converted directly on entry into the cell to protein aspartate. This is unlikely to be so in T. aureurn for the specific activity of aspartate in protein is very much higher than those amino acids which become labelled (Table 2). Further, while the indications are that lysine cannot be synthesized within the sporangia, nevertheless it is incorporated into protein and one can only assume the amino acid is from a supply originally external to the organism. Coleman (1967) presented evidence that Enfodiurn caudafurn Stein engulfed Escherichia coli labelled with individual 14C amino acids and incorporated them into Protozoa protein without apparent prior conversion to other amino acids. Thus it would seem that, while lysine is not synthesized in T. aureurn indicating the partial or total absence of either the

F. M. Paton and D. H. Jennings

475

DAP or AAA pathway, such that label from either L - [ ~ - ' ~ C ] - We wish to thank the late Dr I. J. Galpin and his colleagues aspartic acid and [2-14C]acetate is not incorporated into in the Department of Organic Chemistry for their help with lysine, it is not out of keeping with what is found in some the amino acid content of sporangia of Thraustochytrium and protozoa that label, nevertheless, is transferred into the amino Dr S. T. Moss for his helpful advice on the manuscript. acids, threonine, glutamic acid, alanine and proline. We must conclude that on the basis of these findings REFERENCES T . aureum does not have either the DAP or AAA pathway for ALDERMAN, D. J., HARRISON, J. L., BREMER, G. B. & JONES, the synthesis of lysine. Whether there might by another E. B. G. (1974). Taxonomic revisions in the marine biflagellate pathway is unproven but unlikely. The evidence just presented fungi: the ultrastructural evidence. Marine Biology 25, 345-357. about lysine nutrition of protozoa would suggest otherwise. BAHNWEG, G. (1979). Studies on the physiology of ThraustoHowever, it should be noted that while the absence of chytriales. I. Growth requirements and nitrogen nutrition of saccharopine dehydrogenase in T . aureum suggests that no Thraustochytrium spp., Schizochytrium sp., japonochytrium sp., part of the AAA pathway is present, it is possible that the later Ulkenia spp. and Labyrinthuloides spp. Veroffentlichungen des lnstituts fur Meeresforschung in Bremerhaven 17,245-268. stages of the DAP pathway may be present. Studies by Onodera & Kandatsu (1974) have shown that the rumen BAHNWEG, G. (1980). Phospholipid and steroid requirements of Haliphthorus milfordensis, a parasite of marine crustaceans and ciliate protozoans, Entodinium and Diplodinium are able to Phytophthora epistomium, a facultative parasite of marine fungi. synthesize lysine from diaminopimelic acid. This suggests that Botanica Marina 23, 209-218. these organisms may have possessed the DAP pathway at BAHNWEG, G. & BLAND, C. E. (1980). Comparative physiology some stage - in their evolutionary history but now are in the and nutriton of Lagenidium callinectes and Haliphthoros milfordensis process of losing the pathway. There might be the same fungal parasites of marine crustaceans. Botanica Marina 23, situation in Thraustochytrium. 689-698. The growth studies were designed to investigate further BAHNWEG, G. & JACKLE, I. (1986).A new approach to taxonomy whether or not Thraustochyhiurn could not synthesize lysine of the Thraustochytriales and Labyrinthulales. In The Biology of and therefore required the amino acid in the growth medium Marine Fungi (ed. S. T. Moss), pp. 131-140. Cambridge, U.K.: Cambridge University Press. and whether some of the final steps of the DAP pathway might be present in the organism. The results are somewhat BORUT, S. Y. & JOHNSON, T. W. (1962). Some biological observations on fungi in estuarine sediments. Mycologia 54, inconclusive. Although efforts were made to design media in 181-193. which the omission of diaminopimelic acid or lysine would BROWN, J. W. & COOKSEY, K. E. (1984). Lysine biosynthesis and prevent growth while presence of either amino acid in the the evolution of pennate marine diatoms. ]ournu1 of Experimental medium would sustain growth, these efforts were met with Marine Biological Ecology 80, 197-206. lack of success. It is possible that, as well as the probable BUETOW, G. & LEVEDAHL, B. H. (1964).Comparative physiology requirement for lysine for growth, there is also a requirement and nutrition of Lagenidium callinectes and Haliphthorus milfordensis. for one or more of the five amino acids omitted from the Responses of microorganisms to sterols and steroids. Annual designed medium, namely histidine, valine, methionine, Review of Microbiology 18, 167-194. phenylalanine and tryptophan. This needs to be investigated. CHAMBERLAIN, A. H. L. (1980). Cytochemical and ultrastructural studies on the cell walls of Thraustochytrium spp. Botanica Marina O n the other hand, it is possible that tryptone and yeast 23, 669477. extract contain a growth factor or factors not supplied in media which were used. Candidates for essential growth COLEMAN, G. S. (1967). The metabolism of the amino acids of Escherichia coli and other bacteria by the rumen ciliate Entodinium factors are phospholipids (Bahnweg, 1980; Bahnweg & Bland, caudatum. lournal of General Microbiology 47, 449-464. 1980) and steroids (Buetow & Levedahl, 1964; Vishniac & DARLEY, W. M. & FULLER, M. S. (1970). Cell wall chemistry and Watson, 1953; Vishniac, 1955). taxonomic position of Schiwchytrium. American Journal of Botany Thus there are some uncertainties about how lysine in 57, 761. Thraustochytrium might originate. While it seems unlikely that DARLEY, W. M., PORTER, D. & FULLER, M. S. (1973). Cell wall the amino acid is synthesized by the AAA pathway, the composition and synthesis via Golgi-directed scale formation in possibility that it can under certain circumstances be the marine eukaryote Schizochytrium aggregatum, with a note on synthesized from exogenous diaminopimelic acid cannot be Thraustochytrium sp. Archiv fur Mikrobiologie 90, 89-106. excluded. Nevertheless, in spite of these uncertainties, the data GOLDSTEIN, S. & BELSKY, M. M. (1964).Axenic culture studies of a new marine phycomycete possessing an unusual type of asexual indicate that lysine cannot be synthesized from aspartate by reproduction. American Journal of Botany 51, 72-78. either of the two pathways normally found in fungi. The GUTTMAN, H. N. (1967). Patterns of methionine and lysine evidence that we have obtained in this study is more in biosynthesis in the Trypanosomatidae during growth. journal of keeping with what we know about Protozoa with respect to Protozoology 14, 267-2 71. the origin of lysine in their cells. In saying that, we are not HORI, H., LIM, B.-L. & OSAWA, S. (1985).Evolution of green plants necessarily proposing any necessary phylogenetic relationship as deduced from 5s rRNA sequences. Proceedings of the National between Thmwtochytrium and those organisms. However, our Academy of Sciences of the United States of America 82, 82&823. findings, when added to those of several other investigations HUYSMANS, E., DAMS, E., VANDENBERGHE, A. & DE referred to in the Introduction concerned with the phylogenetic WACHTER, R. (1983).The nucleotide sequences of the 5s rRNAs relationships of Thraustochytrium and related organisms, of four mushrooms and their use in studying the phylogenetic enhance that view that they are not related to the fungi. position of basidiomycetes among the eukaryotes. Nucleic Acids Research 11,2871-2880.

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