Leucine uptake by enterobacterial and algal members of larval anuran gut flora

Camp. Biochem. Physiol. Vol. 101B, No. 4, pp. 527-530, 1992 Printed in Great Britain

0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press pie

LEUCINE UPTAKE BY ENTEROBACTERIAL A N D ALGAL MEMBERS OF LARVAL A N U R A N GUT FLORA TREVOR J. C. BEEBEEand ADELINE L.-C. WONG Department of Biochemistry, University of Sussex, Falmer, Brighton BN1 9QG, U.K. (Tel: 0273 606755) (Received 6 September 1991) Abstract--1. We have measured leucine uptake by purified enterobacterial and heterotrophic algal (genus Prototheca) cells isolated from the intestines of anuran larvae. 2. Prototheca cells had a ten-fold greater Kmthan enterobacteria for leucine uptake, but Vmx was orders of magnitude higher in the algal cells on per cell, per cell volume or per cell surface area bases. 3. Leucine incorporation was copper-sensitive in the algae but not in the bacteria. 4. Mixed populations of algae and enterobacteria were competitively equal with respect to leucine incorporation at limiting concentrations of the amino acid.

INTRODUCTION Although enterobacteria are dominant members of the gut flora in most vertebrates, circumstances also exist in which other taxa become equally or more abundant. During crowded growth of anuran amphibian larvae, for example, large numbers of unpigmented, heterotrophic algae (Prototheca species) are present in the gut and cause growth inhibition in the smaller tadpoles of mixed populations (Richards, 1962; Beebee, 1991). Protothecans are a poorlystudied group of unicellular organisms related to green algae of the Chlorococcales family (Huss et al., 1988; Huss and Sogin, 1990) and with four known species (Pore, 1985), though that inhabiting the larval anuran gut is immunologically distinct and probably novel (Wang and Beebee, submitted). We set out to investigate how these highly specialized eukaryotic heterotrophs, with cell cycle times > 20 times longer than those of the enterobacteria and an apparent requirement for gut passaging to maintain their division cycle, compete successfully in the intestine for available nutrients. Protothecans are generally associated with organic wastes of various kinds, and are capable of causing disease in M a n and animals under certain circumstances (Pore et al., 1983; Pore, 1985). Little is known about their biochemistry or their relationships with other organisms, especially the bacteria with which they would appear to be in direct competition. MATERIALS AND METHODS Materials Tadpoles of Rana temporaria and Bufo calamita, separately or mixed, were grown in tanks containing 1-1. dechlorinated tapwater and under a standardised feeding regime (Beebee, 1991). Enterobacteria were isolated from Rana temporaria larvae following dissection of gut sections, gentle disruption with a dounce homogeniser and plating on afar with L-broth using standard microbiological techniques (Maniatis et al., 1982). After serial dilution, individual colonies were grown in 20 rnl batches of L-broth at 25°C and harvested when absorbance at 550 nm reached approximately 0.2 (i.e. at

5 X 1 0 7 cells/ml). Cells were pelleted at 2000 g, washed in M9 salts and resuspended in M9 for use in assays. Protothecans were isolated from tadpole faeces using differential filtration, centrifugation and percoll density gradients as described elsewhere (Beebee, 1991). (3H)-Leucine, (5.7TBq/mmol), NCS tissue solubiliser and OCS scintillation fluid were from Amersham International (Amersham, U.K.). Percoll was from Pharmacia (Milton Keynes, U.K.); and routine chemicals were all from BDH (Poole, U.K.) and Sigma (Poole, U.K.). Enterobacteria and Prototheca estimations One-centimetre sections of tadpole intestines were homogenised in 0.25 ml L-broth, after which a series of serial dilutions were made, plated onto L-broth afar and incubated overnight at 25°C. All platings were duplicated, and colony counts were used to quantify enterobacterial numbers. Samples of homogenates were used to estimate Prototheca numbers directly by haemocytometer counting, again with duplicate counts for each sample. Leucine incorporation Assays were set up on ice, normally in final volumes of 100/zl and always in duplicate, with final salt concentrations at 1 x M9 (Maniatis et al., 1982). Each assay also contained 0.2% (w/v) glucose and 37 kBq (3H)-leacine, other components as specified by individual figure legends, and normally either 104 Prototheca or 107 enterobacteria. Incubation was for 30min at 25°C, after which I ml 10mM leucine was added to each assay on ice, cells pelleted by centrifugation at 13,000g for 5rain (microfuge), resuspended in 1 ml 10 mM ice-cold leucine and pelleted again. Final pellets were resuspended in 50#1 distilled water, dispensed onto GF/C discs, dried and radioactivity estimated by scintillation counting. Gradient analysis of mixed-cell populations Large-scale assays (150 #1 with 5 x l0 s enterobacteria, 106 protothecans or both) were carried out as described above as far as resuspension in 50#1 distilled water. These cell suspensions were then overlaid onto 11 m148% (v/v) percoll gradients preformed by centrifufation in an angle rotor at 43,000g for 30rain and containing 0.1 M NaC1. The gradients were then centrifuged at 450 g for 30 rain, fractionated into 10 l-ml aliquots and 0.2 ml of each fraction incubated with Imt NCS tissue solubiliser for I hr at 50°C. After neutralisation with 30#1 glacial acetic acid the samples were mixed with 10 ml OCS scintiUant and radioactivity estimated in the usual way. 527

528

TREVORJ. C. B~n~ and AD~IN~ L.-C. WoNo Table 1. Enterobacteria and Prototheca in tadpole gut sections lk(5-)

Tadpole growth conditions Bc (10-) Rt ( 5 - ) Rt (10-) Rt (5+)

Bc (5+)

Body length (ram)

17 (< 1)

16 (< 1)

26 (3)

25 (< 1)

27 (< 1)

13 (< I)

Prototheca (cells/0.4 mm gut)

36 (7)

29 (6)

155 (45)

370 (5)

128 (14)

207 (199)

Enterobacteria (cells/0.4 mm gut)

1550 (1202)

1250 (353)

800 (424)

0 (0)

3600 (1697)

1650 (1768)

0

28

Entero/Proto

43

44

5

8

Tanks contained 5 or 10 hatchling (10 mm) B. calamita larvae (Be5-, 10-), 5 or 10 well-grown (>20ram) R. temporaria larvae (Rt5-, 10--) or 5 larvae of each species mixed together (5 +). After 8 days of a standard feeding regime (100 mg vegetable pellets/tank every 2 days) animals were sacrificed and gut flora quantified as descried in Methods. Data are body sizes at end of experiment and numbers of cells from duplicate estimations (2 separate tadpoles from each treatment) with standard deviations in parentheses.

RESULTS

Tadpoles grown under various conditions were analysed for gut microorganisms as shown in Table 1. At the densities of animals used, both enterobacteria and protothecans were abundant and in terms of cell mass (Prototheea are approximately 65-fold larger than enterobacteria by volume) the algae were often the dominant organism present.

? 0

A

o

x ~

o

~° I,J

0

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i

)

I

i

2

4

S

S

10

CELL No. x lO-4(Pt} or xlO-7(Et)

? ~

B

x

8 -~® E

12

24

36

48

60

TIME (MIN}

Fig. l. Leucine incorporation by gut microorganisms. A: Assays contained varying numbers of enterobacteria (A) or protothecans (~lr) prepared and incubated as described in methods. B: Assays contained either l08 enterobactefia (A) or l05 prototheca3s (11) each in final volumes of ].0 rid; duplicate 100/~l aiiquots were removed at various times

during incubation at 25°C for assessment of (3H)-Ieucine incorporation. Error bars show ranges of duplicates.

There was a general trend for numbers to be inversely related, and certainly the tadpoles with the highest numbers of protothecans (Rana temporaria larvae grown at 10/1) had few, and sometimes no, detectable enterobacteria in the gut sections analysed. Both cell types incorporated leucine actively under the assay procedure used, with incorporation related to cell number and continuing for at least 60 min at 25°C as shown in Fig. 1. Brief heat treatment (80°C for 5 rain) prior to assay abolished all incorporation by both the prokaryote and the eukaryote. However, there were substantial differences between the two cell types with respect to the properties of their leucine transport systems. That of enterobacteria had a relatively high affinity for leucine, with a Km of 2/~M or less, but exhibited a Vm~ much lower than the system operative in prototheca (Fig. 2 and Table 2). Protothecans took up much more leucine than enterobacteria under these assay conditions (and others tested) whether this was measured as a function of cell number (>3000-fold difference), cell volume (>80-fold difference) or cell surface area ( > 300-fold difference). The Km of uptake in Prototheca was, however, an order of magnitude higher than that observed in the bacteria, indicative of a transport system with lower affinity for substrate operating most efficiently at relatively high substrate concentrations. Algae, including Prototheca, are particularly susceptible to copper toxicity (Richards, 1962; Beebee, 1991). This was reflected in the sensitivity of (3H)leucine incorporation to copper sulphate, which in Prototheca was inhibited over the toxically-active range but which in enterobacteria was unaffected even at high concentrations (Fig. 3). Finally, we investigated uptake of limiting concentrations of leucine by the two cell types alone or mixed together, separating them after uptake of radioactivity by centrifugation through percoll density gradients. As shown in Fig. 4, reduced incorporation in mixed populations was about equally spread between the bacteria and algae; in both cases the decline relative to unmixed cells was around 33-35%. There was therefore no evidence that under the conditions tested either cell type was able to exploit limiting concentrations of leucine at the expense of the other.

Leucine uptake by anuran gut flora

~

I 0

A

,! 8-

529

o

8O. O

z

o~ 0

I 20

10

I 30

I 40

O3 50

E

0

0.1

LEUCINE CONC. (pM)

1

10

100

1000

CuSO4 CONC. [/Jg/ml)

o

Fig. 3. Copper sensitivity of (3H)-lcucine incorporation.

-

13

Duplicated assays were as described in Methods, with various concentrations of copper sulphate present throughout. (ll), enterobacteria; (A), protothecans.

o

0

4

S

12

.) 20

16

1IS Fig. 2. Substrate concentration-dependence of leucine incorporation. Duplicated assays were as described in methods, each in final volumes 50 #1 with 37 KBq (3H)-leucine and various concentrations of unlabelled leucine. Enterobacteria (A) and protothecans (ll). DISCUSSION

The very high rates of leucine uptake by protothecan cells relative to bacteria was a striking and unexpected finding. Multiple transporter systems exist for the uptake of amino acids by both prokaryotic and eukaryotic cells, including mechanisms based on active transport and facilitated diffusion (Bender, 1985; Ames, 1986; Yudilevitch and Boyd, 1987). The experiments reported here gave only a preliminary indication of how the processes differ between two evolutionary distant but ecologically similar heterotrophic organisms that share the environment of the larval amphibian gut. The properties we have measured may differ according to assay conditions and in particular according to endogenous amino acid pool sizes in the cells under study, although we have looked at bacteria and Prototheca from several different isolates and after various periods of preincubation without added nutrients (data not shown), all of which gave results similar to those reported above. We have no information on the species diversity of Table 2. Kinetic parameters of leucine uptake Km Vmax : p m o l / 3 0 rain/100,000 cells

pmol/30rain/100,000#m3 cell volume pmol/30rain/100,000#m2 cell surface area

Enterobacteria

Protothcca

2#M

15-20#M

0.15

500.00

0.09

7.70

0.02

6.37

amphibian gut enterobacteria, though colonies and individual cells grown on L-broth plates were apparently homogeneous with respect to both morphology and growth characteristics. We are therefore confident that our comparative study has employed the most abundant gut bacterium in the larvae, but cannot rule out the presence of other types with different nutrient-assimilating properties. It looks as if prothecans are particularly adept at sequestration of large amounts of amino acids during their passage through the digestive system, a feature which is compatible with other observations of this organism's life cycle. Protothecans of the type inhabiting tadpole intestines swell during gut passage and accumulate electron-dense storage materials in large vacuoles, which in turn prepares them for at least one round of cell division before they require (as obligate parasites) a further gut passage to replenish nutrients. Enterobacteria replicate continuously inside the gut and O3 I

o

z

o

~2

FRACTION No. (FROM TOP)

Fig. 4. Leucine sequestration was not competitive between enterobacteria and protothecans. Three assays were set up, each in final volumes of 150#1 and with standard components including 111 KBq (3H)-leucine, and also containing 5 x 10s enterobacteria alone (Ill), 104 protothecans alone (A), or the same amounts of both cell types mixed together (V). After incubation at 25°C for 30 rain the cells were washed twice, layered over preformed percoll density gradi: ents and centrifuged, all as described in Methods. Gradients were fractionated into 11 x l-m1 aliquots, 0.2 ml from each of which were used for radioactivity estimation.

TREVORJ. C. BEF..BEEand ADELINEL.-C. WON6

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are efficient scavengers of low concentrations of amino acid substrates. It may be that the protothecans sequester most of their nutrients at an early stage during gut passage, when concentrations are high prior to absorption by competing villi and enterobacteria, but the inability of the latter to fare relatively better than protothecans at low amino acid concentrations in vitro (Fig. 4) was surprising considering the kinetic properties summarised in Table 2. Presumably, large numbers of low-affinity transporters offset the inherent advantage of the highaffinity bacterial ones simply because the latter were present at lower frequency, but evidently this and the incorporation of other nutrients by these organisms requires further comparative studies. Acknowledgements--We thank the Natural Environment Research Council for financial support. REFERENCES

Ames G. F. L. (1986) Bacterial periplasmic transport systems: strucutre, mechanism and evolution. A. Rev. Biochem. 55, 397-425.

Beebee T. J. C. (1991) Purification of an agent causing growth inhibition in anuran larvae and its identification as a unicellular unpigmented alga. Can. J. Zool 64, 2146-2153. Bender D. A. (1985) Amino Acid Metabolism (2nd Edn). John Wiley & Sons, New York. Huss V. A. R. and Sogin M. L. (1990) Phylogeneticposition of some Chlorella species within the Chlorococcales based upon complete small-subunit ribosomal RNA sequences. J. molec. Evol. 31, 432-442. Huss V. A. R., Wein K. H. and Kessler E. (1988) Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella. Arch. Microbiol. 150, 509-511. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. Pore R. S. (1985) Prototheca taxonomy. Mycopathologia 90, 129-139. Pore R. S., Barnett E. A., Barnes W. C. and Walker J. D. (1983) Prot0theca ecology. Mycopathologia 81, 49-62. Richards C. M. (1962) The control of tadpole growth by alga-like cells. Physiol. Zool. 35, 285-296. Yudilevitch D. L. and Boyd C. A. R. (1987) Amino Acid Transport in Animal Cells. Manchester University Press, Manchester.