- Email: [email protected]
Microbial production and degradation of γ-aminobutyric acid (GABA) in the abalone larval settlement habitat
ELSEVIER
FEMS Microbiology Ecology 17 (1995) 205-212
Microbial production and degradation of y-aminobutyric ( GABA) in the abalone larval settlement habitat H.F. Kaspar
*,
acid
D.O. Mountfort
Cawthron Institute, Pricate Bag 2, Nelson, New Zealand
Received 12 December 1994; revised 19 April 1995; accepted 19 April 1995
Abstract By producing or degrading gamma-aminobutyric acid (GABA), microbes may affect the settlement of abalone (Haliotis) larvae. GABA was not detectable in extracts of crustose red algae (CRA) which are the preferred settlement substratum for Huliotis larvae. The CRA surfaces and their associated biofilms did not produce GABA from glutamate, and GABA production from putrescine required gabaculine (transaminase inhibitor). The GABA-degrading activity on the CRA surfaces was removed by surface-sterilisation. It ranged from 0.45 to 1.12 nmol cm * h-’ with insignificant seasonal variation. Removal of grazing molluscs from CRA-covered stones led to the growth of a visible biofilm and markedly increased GABA degradation in 2 weeks. Within a few days of placing clean, sterile glass surfaces in the settlement environment, biofilms with GABA-degrading activity developed, and after 11 weeks incubation the GABA degradation rate on glass marbles was 0.37 nmol cmP2 h- ‘. GAB A was not completely oxidised, and the products of GABA metabolism showed similar chromatographic behaviour to alanine and glutamate. In the typical New Zealand Huliotis settlement habitat, GABA
is not likely to play a significant role as an inducer of abalone larval settlement because on the preferred settlement surfaces it is not readily produced from its precursors but it is easily degraded. Keywords: Invertebrate larval settlement; Marine bacteria; Microbial metabolism; Biofilm
1. Introduction Gamma-aminobutyric acid (GABA) inhibits the velar cilia movement in planktonic abalone (Huliotis) larvae, thus promoting their settlement [1,2]. In abalone hatcheries this trait is successfully used to settle competent larvae onto the tank surfaces [3], and a similar role of GABA in the natural Huliotis settlement habitat may be proposed. Marine bacteria
* Corresponding author. Tel: +64-3-548-2319; Fax: +64-3546-9464; Email: [email protected]
can produce GABA from amines or amino acids [4,5], and the ability to degrade GABA is common among marine heterotrophic bacteria [6]. Therefore, marine microorganisms may affect the settlement of Haliotis larvae by affecting the GABA concentration in the settlement environment. The aim of this study was to describe the dynamics of GABA on natural Huliotis settlement surfaces in order to evaluate the role of GABA as a natural settlement inducer. Production and consumption of GABA on the hard surfaces of the coastal marine abalone settlement habitat, particularly on crustose red algae (CRA), are described. GABA was only produced
0168.6496/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SsDl 0168-6496(95)00025-9
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H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
from putrescine in the presence of a transaminase inhibitor, but constitutive GABA-degrading activity in microbial films on settlement surfaces was common. It is unlikely that GABA plays a significant role in Haliatis larval settlement in typical New Zealand Haliotis habitats.
2. Materials
and methods
2.1. GABA analysis GABA was derivatised with phenylisothiocyanate and chromatographed by HPLC [7]. The identity of the GABA peak was established by co-chromatography of the sample mixed with a standard. GABA was measured by peak height comparison with standards. The detection limit for GABA was 0.1 FM. Samples containing radioactive GABA were analysed by the same procedure, except that the injection volume was increased from 4 ~1 to 50 ~1. At an elution rate of 1 ml min-‘, fractions of 0.5 or 1 ml were collected from the detector outlet. These were counted with a Beckman LS 3801 in toluene-based scintillant with Triton-X-100 according to previously published procedures [8]. For the measurement of residual radioactivity, 0.5 ml methanol was added to 0.5 ml sample which was then evaporated to dryness. The residual radioactivity was thus associated with non-volatile compounds. 2.2. Study site and natural GABA concentrations A tidal rock pool in Cable Bay, Nelson, was chosen for the study. The pool was near the low tide level and provided an easily accessible sample of rocky subtidal, the typical habitat of all three New Zealand Haliotis species. The pool contained small stones covered by crustose red algae (mainly Lithothamnion sp.). The solid rock sides were colonised by crustose and articulate corallines (Lithothamnion sp., Corallina sp.) and a variety of foliose seaweeds (Hormosira sp., Carpophyllum sp., Porphyra sp.). Small stones covered by crustose red algae were collected from the tidal rock pool. They were then shaken at 25°C in a small amount of HCl(O.O5-5 N) for l-30 min, to allow for the selective extraction of
Ecology I7 (1995) X-212
amino acids, ranging from those dissolved in the tissue to those bound in peptides. Extracts were analysed for amino acids as above. 2.3. ‘H-GABA -‘H-glutamate
production
from
‘H-putrescine
and
Freshly collected stones (l-2 cm diameter, > 90% surface covered with crustose red algae) were transported in seawater to the laboratory and then placed in duplicate groups of 6-8 in 250-ml beakers, each containing 60 ml of seawater collected from the same area. This water had previously been amended with unlabelled putrescine or glutamate (50-1000 PM) and 1,4 3H-putrescine (0.4 &i ml-‘, specific activity 22.9 Ci mmol- ’ ) or L-[G-3 HIglutamate (0.5 PCi ml-‘, specific activity 50 Ci mmoll ’ 1. Gabaculine (10 pug ml - ‘, inhibitor of GABA transaminase) was added to some beakers, and controls with formalin (0.4% v/v) or without stones were set up. Beakers were incubated at 25°C on a transversal shaker at 120 ‘pm. At various intervals over a 120 h period, 1 ml water samples were removed and stored at - 18°C for later analyses of GABA and radioactivity. At incubation times > 90 h, bacterial growth was observed, and samples were centrifuged at 6000 X g for 15 min at 2°C. Supernatants were stored at - 18°C until they were analysed. 2.4. Measurement
of GABA degradation
Flat, coralline covered stones of l-4 cm diameter were collected from the same rock pool as above. The coralline thalli were polished off the edges of the stones so that the areas of coralline cover could be measured accurately. The stones were separated in four groups, giving total coralline areas of 73-86 cm7 per group. Each group was placed in a 150-ml beaker and covered with seawater (56-69 ml) containing 10 FM GABA. The stones were then incubated at the bulk water temperature in the area where the stones had been collected. Samples (0.5 ml) were taken over a 7-10 h period and analysed for GABA. To determine the effect of season on the GABA degradation rate, the stones were returned to the rock pool immediately after the experiment. They were retrieved at intervals of several weeks during the following year, and the experiment was repeated.
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology Table 1 Seasonal variation of GABA degradation rates on crustose red algal surfaces of small stones in a rock pool at Cable Bay, Nelson Date
7.9.90 3.10.90 16.11.90 3.1.91 27.2.91 15.4.91 12.6.91
Rock pool temperature (“C)
14 16 20 16 27 22 14
a
Experiment temperature (“C)
14 14 19 20 22 22 15
a
Rate +95% conf. interval b (nmol cm-* h-l)
1.10+0.20 0.70 f 0.25 1.07rt 0.07 0.45 rto.04 0.82 F 0.21 1.05rto.19 1.12k0.56
a Depending on the tide and the weather, the water temperaturein the rock pool when the stones were collected was several degrees higher or lower than the subtidal bulk water temperature. The experiments were carried out at the temperature which prevailed in the subtidal on the days before the stones were collected. b Four replicates.
Immediately before these repeats the non-coralline stone surfaces were wiped with acetone. Dates and temperatures for each repeat experiment are given in Table 1. Duplicate control beakers containing no stones, bare stones, or coralline-covered stones which had been wiped with acetone were incubated along with the beakers containing the coralline-covered stones to determine the location and nature of the GABAdegrading activity. To determine the effect of grazing on the rate of GABA degradation, stones (l-2 cm diameter) covered with coralline thalli and various small grazing molluscs were collected from the pool as above. Stones and grazers were brought to the laboratory and kept together in an aquarium until the following day, when the grazers were removed, and the stones divided into 4 groups of 15 stones with similar total surface area. Each group was incubated in 50 ml of seawater containing 50 PM GABA at 23°C on a transversal shaker at 120 ‘pm. Samples (0.1 ml) were taken at intervals of 0.5-l h and analysed for GABA. After 8 h the stones were washed with fresh seawater. Two groups each were placed in 4 1 of aerated seawater supplemented with NaNO, (250 PM), NaSiO, (25 ,uM) and KH,PO, (12 PM). To one aquarium the following grazers were added (numbers in brackets): Diloma nigerrima (4), Melographia aethiops (l), Sypharochiton pelliserpentis (1) and
207
Ecology 17 (199.5) 205-212
Cellana radians (2). The aquaria were exposed to the natural light fluctuations and to temperatures fluctuating between 20 and 24°C. The measurement of GABA degradation was repeated after 7, 15 and 22 days. 2.5. Development
of GABA-degrading
biofilms
Glass marbles (15 mm diameter) were washed with detergent and heat-sterilized. Groups of 16 marbles in plastic netting and individual marbles were placed in a tidal rock pool at Cable Bay. At intervals of several days, marbles were retrieved. Duplicate groups of 8 marbles were incubated in 35 ml seawater containing 10 /.LM GABA at 25°C on a transversal shaker at 120 rpm. Samples (0.5 ml) were taken at intervals and analysed for GABA.
3. Results and discussion 3.1. Natural GABA concentration Extraction of crustose red algae with HCI liberated a complex spectrum of amino acids, but no GABA was found in any sample. The natural GABA pool was therefore below detection limit (0.1 FM or 30 nmol cme2 of crustose red algal surface). This confirms results of other researchers [9]. However, the low GABA concentrations in bulk extracts do not mean that the natural GABA concentration on surfaces is too low to affect abalone larval behaviour. 3.2. Production 3H-glutamate
of 3H-GABA from 3H-putrescine and
GABA has previously been shown to be produced from glutamate and putrescine by isolates of marine bacteria [4,5], but there has been no documentation of the production of the compound at the sites of Huliotis larval settlement. In the absence of gabaculine, incubations of CRA-covered stones with 3Hputrescine and 3H-glutamate (0.05-l mM) resulted in little (< 1 PM), or no GABA production over a 120 h time-course, respectively. In the presence of gabaculine, GABA was produced from labelled putrescine as indicated from the appearance of label (> 90%) in the 6.0 to 6.5 min
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
208 3000
-
A 2500 2000
E.1500
cl
-
I I
1000 t
500
Oh
” yl 9 u)
0
U-J
b
d
d
i
d
B
d
d
d
d
Retention
time (min)
I
0
20
40
60
80
100
Time(h)
Fig. 1. A: Profile of 3H-GABA during HPLC of 50 ~1 of sample taken from incubation of 3H-putrescine (1 mM) in the presence of CRA-stones and gabaculine after 96 h. B: Time-course for the production of GABA from 1 mM putrescine in the presence of CRA-stones and gabaculine. Symbols: A, GABA (determined directly by HPLC); v , dpm of GARA per ml of incubation; 0, GARA determined from counts and the specific activity of the starting substrate @Act, 1 X lo6 dpm pmol- ‘).
fraction of a chromatographic run (Fig. lA), corresponding to the retention time of the GABA standard of 6.2 min. No GAEIA was produced from glutamate ( < 0.5 PM). GABA production determined from the counts in the 6.0-6.5 min fractions, and the specific activity of the labelled substrate closely matched that
Ecology I7 (1995) 205-212
determined directly by comparison of peak heights with GABA standard as shown for incubations with 1 mM putrescine (Fig. 1B). The highest GABA concentration resulting from putrescine degradation was 70 PM, which was obtained after 96 h incubation. At starting levels of putrescine of less than 1 mM, maximal levels of GABA did not exceed 45 ,~LMat any stage of the time-course. In controls with stones omitted, or with formalin added, there was no production of GABA. These results indicate GABA as an intermediate, rather than a product of putrescine degradation by microorganisms residing on CFL4-covered surfaces. Although production of GABA from putrescine and glutamate has previously been demonstrated in pure cultures of marine microorganisms [4,5], our experiments showed that GABA accumulated only in the presence of putrescine and the transaminase inhibitor, gabaculine. Several possibilities may explain the failure to detect GABA from glutamate. The first is that label from glutamate would not be incorporated into GABA if it were produced from transamination since glutamate was tritiated only in carbon positions 2 to 4. GABA produced in this way would only utilise the amino group of glutamate, and its formation would be inhibited in the presence of gabaculine. If GABA were produced from decarboxylation of glutamate [lo] then its accumulation would be expected through inhibition of the first step in its utilisation in the presence of gabaculine. This also did not occur. Thus decarboxylation appears unlikely as the means of glutamate conversion to GABA. The conditions under which significant GABA production from putrescine occurred in the presence of the blocking agent, gabaculine, are unlikely to be encountered in the natural coastal environment, and therefore unlikely to operate in the settlement of abalone larvae. It seems more likely that in the natural environment, the presence of the compound is controlled by the enzymes involved in its utilisation. The finding that GABA did not accumulate in the absence of inhibitor suggests that these enzymes effectively remove any GABA that is produced. 3.3. Location, microbial GABA-degrading activity
nature
and products
of
GABA disappeared from water which was incubated in the presence of stones which appeared bare
H.F. Kaspar, D.O. Mountfort/
FEMS Microbiology
209
Ecology I7 (1995) 205-212
replicates was often high, so that there was no significant (P = 0.05) seasonal effect on the GABA degradation rate of the CRA surfaces (Table 1). The rock pool temperatures measured when the stones were retrieved ranged from 14°C to 27°C. The actual temperature range was substantially wider due to the extremes of hot summer days and cold winter nights. However, because the pool was submerged for most of the time, the experiments were carried out at the more significant bulk water temperatures (14-22°C).
0
I
’ 0
1
2
3
4
5
<\r 6
7
Time(h)
Fig. 2. Location and nature of GABA-degrading activity. Stones were incubated as described in Materials and methods with GABA at an initial concentration of 10 PM. Symbols: 0, bare stones; +, CRA covered stones; n , water; A, acetone-wiped stones.
or were covered with crustose red algae, but not from water only, or water incubated in the presence of stones which had previously been wiped with acetone (Fig. 2). GABA degradation was therefore carried out by microbes colonising hard surfaces, regardless of whether these surfaces were covered by crustose red algae. Such surfaces are the preferred settlement substrata for abalone larvae [ll-131 and the presence of GABA-degrading activity on these surfaces reduces the likelihood of GABA playing a role in abalone larval settlement. Stones covered in crustose red algae were incubated in seawater containing 10 PM GABA and 0.2 nM 3H-GABA (0.018 &i ml-‘). Samples were taken at intervals for the measurement of GABA, residual radioactivity and radioactive GABA. While GABA and radioactivity in the GABA peak disappeared within a few hours, 63% of the initial 3H appeared in fractions containing glutamate and aspartate. Thus, GABA was largely converted into amino acids which are not inducers of abalone larval settlement ([14], Berkett et al., manuscript in preparation). 3.4. Seasonal [lariation of GABA degrading on crustose red algae
3.5. Effect of grazing on GABA degradation on crustose red algal surfaces
activity
The GABA degradation rate was compared between CRA-covered stones which were either exposed to grazers such as chitons or were free of grazers. During the entire experiment (22 days) the grazed stones remained pink with a clean CRA surface. On the light side of the ungrazed stones a brown diatom film had developed after 7 days. After 15 days small Enteromorpha filaments were visible, and after 22 days these filaments reached 8 mm, and the diatoms had receded. The CRA were covered by a slime. For the first 7 days, GABA degradation was slow, and there was no significant difference in the GABA-degrading activity between grazed and ungrazed stones. Following this initial period, the GABA degradation rate increased on the ungrazed surfaces while it remained low on the grazed surfaces (Fig. 3). Grazing prevented the overgrowing of CRA surfaces by microbial films and thus kept the GABA degrading activity constant and low. The frequent scouring of hard surfaces by grazers appears to have a more significant effect on the GABA-degrading activity than the seasonal temperature changes which are modified by strong tidal temperature fluctuations (Table 1 and Fig. 3). 3.6. Development young biofilms
of GABA-degrading
activity
in
activity
The GABA degradation rates ranged from 0.45 to 1.12 nmol cm-’ h- ‘, but the variability between
GABA-degrading activity can be constitutive in marine bacteria [6]. In order to demonstrate that GABA-degrading activity is constitutive in young marine biofilms, we placed clean, sterile marbles in a
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
210
tidal rock pool and measured the GABA-degrading activity at intervals of several days. With freshly sterilised marbles, the GABA degradation rate was 30 pmol cme2 h-‘. This activity was assumed to be in the water, and subsequent measurements were corrected for it. Within a few days of placement in the tidal pool, the marbles had a measurable GABA degradation activity (Fig. 4). This activity reached 131 pmol cm-* h-* in 23 days on the marbles which were placed in the pool individually, and 350 pmol cmm2 h-’ i n 77 days on the marbles which were placed in the pool as a group in plastic netting. Probably due to less abrasion, the rate was always higher on the marbles in plastic netting than on the free marbles, many of which were lost so that this
r
::p=
3 $
400
7 _c ys 300 -a $ E p 200 m $ d E 100 5 0 0
IO 20
30
40
50 60
70
80
Time (days)
Fig. 4. Degradation of GABA in young biofilms. GABA was determined in subincubations (1 to 8 h) with marbles taken from tidal rock pool at different intervals over 80 days. Symbols: n , individual marbles; 0, marbles placed in plastic netting.
0 Days
60
Ecology I7 (1995) 205-212
experiment had to be terminated after 23 days. Macro-algal growth and tubeworm deposits developed on the marbles after 20 days and were scraped off before the incubation in presence of GABA, so that the measured rates related to thin microbial biofilms only and were likely to be lower than the total rate. The lower rates on marbles (Fig. 4) compared to CRA-covered surfaces (Table 1) may be a reflection of the pitted CEU surface, where microbes can escape the grazing in conceptacles 19,151.
60 r
3.7. Conclusion
22 Days
0
1
2
3
4
5
6
7
8
Time(h)
Fig. 3. Effect of grazing on GABA-degradation on crustose red algal surfaces. GAE%A was determined in subincubations (1 to 8 h) with stones taken from aquaria at different intervals over 22 days. Symbols: 0, stones exposed to grazers; 0, ungrazed stones (means and standard deviations of duplicates).
Our studies and those of others [9] have failed to demonstrate GABA in coralline algae. The microbial communities on CRA surfaces produced GABA from putrescine only in presence of the GABA transaminase inhibitor gabaculine and did not produce GABA from glutamate. GABA was degraded by the microbial community on the hard surfaces of a typical New Zealand abalone settlement environment. Biofilms with constitutive GABA-degrading activity developed rapidly on clean surfaces. GABA degraders were present on CRA-covered stones yearround and withstood grazing by various molluscs. These results, together with our earlier published data on GABA production and degradation by marine bacteria suggest that GABA is unlikely to play a
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology Ecology 17 (1995) 205-212
significant role as an inducer of abalone larval settlement in a typical New Zealand Haliotis habitat.
Acknowledgements We thank our Cawthron colleagues for valuable discussions. Maggie Atkinson and Jan Holland provided excellent technical assistance. This work was part of contract CAW202 with the New Zealand Foundation for Research, Science and Technology and supported by the New Zealand Lottery Board.
References [l] Morse, D.E. (1992) Molecular mechanisms controlling metamorphosis and recruitment in abalone larvae. In: Abalone of the World: Biology, Fisheries and Culture (Shepherd, S.A., Tegner, M. and Guzman Del Proo, S.A., Eds.), pp. 107-109. Fishing News, Oxford. [2] Morse, D.E., Hooker, N., Duncan, H. and Jensen, L. (1979) Gamma aminobutyric acid a neurotransmitter induces planktonic abalone larvae to settle and begin metamorphosis. Science 204, 407-410. [3] Searcy-Bernal, R., Salas-Garza, A.E., Flores-Aguilar, R.A. and Hinojosa-Rivera, P.R. (1992) Simultaneous comparison of methods for settlement and metamorphosis induction in the red abalone (Haliotis rufescens). Aquaculture 105, 241250. [4] Mountfort, D.O. and Pybus, V. (1992) Effect of pH, temperature and salinity on the production of gamma aminobutyric
211
acid (GABA) from amines by marine bacteria. FEMS Microbiol. Ecol. 101, 237-244. [5] Mountfort, D.O. and Pybus, V. (1992) Regulatory influences on the production of gamma-aminobutvric acid by a marine pseudomonad. Appl. Environ. Microbial. 58, 237-242. [61 Kaspar, H.F., Mountfort, D.O. and Pybus, V. (1991) Degradation of gamma aminobutyric acid (GABA) by marine microorganisms. FEMS Microbial. Ecol. 85, 313-318. R.L. and Meredith, S.C. (1984) Amino acid [71 Heinrikson, analysis by reverse-phase high-performance liquid chromatography: precolumn derivatisation with phenylisothiocyanate. Anal. Biochem. 136, 65-74. b31Mountfort, D.O. and Roberton, A.M. (1978) Origin of fermentation products formed during the growth of Bacteroides ruminicola on glucose. J. Gen. Microbial. 106, 353-360. A.L. (1991) [91 Johnson, C.R., Muir, D.G. and Reysenbach, Characteristic bacteria associated with surfaces of coralline algae: a hypothesis for bacterial induction of marine invertebrate larvae. Mar. Ecol. Progr. Ser. 74, 281-294. [lOI Streeter, J.G. and Thompson, J.F. (1972) In vivo and in vitro studies on y-aminobutyric acid metabolism with the radish plant (Raphanus satious L.1. Plant Physiol. 49, 579-584. and [ill Morse, A.N.C. and Morse, D.E. (1984) Recruitment metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae. J. Exp. Mar. Biol. Ecol. 75, 191-215. 1121 Moss, G.A. and Tong, L.J. (1992) Effect of stage of larval development on the settlement of the abalone, Haliotis iris. N.Z. J. Mar. Freshwater Res. 26, 69-73. 1131 Moss, G.A. and Tong, L.J. (1992) Techniques for enhancing larval settlement of the abalone, Haliotis iris, on artificial surfaces. N.Z. J. Mar. Freshwater Res. 26, 75-79. [I41 Morse, D.E., Hooker, N. and Duncan, H. (1980) GABA induces metamorphosis in Haliotis, V: stereochemical specificity. Brain Res. Bull. 5, 381-387. on surfaces of b51 Kaspar, H.F. (1992) Oxygen conditions coralline red algae. Mar. Ecol. Prog. Ser. 81, 97-100.
FEMS Microbiology Ecology 17 (1995) 205-212
Microbial production and degradation of y-aminobutyric ( GABA) in the abalone larval settlement habitat H.F. Kaspar
*,
acid
D.O. Mountfort
Cawthron Institute, Pricate Bag 2, Nelson, New Zealand
Received 12 December 1994; revised 19 April 1995; accepted 19 April 1995
Abstract By producing or degrading gamma-aminobutyric acid (GABA), microbes may affect the settlement of abalone (Haliotis) larvae. GABA was not detectable in extracts of crustose red algae (CRA) which are the preferred settlement substratum for Huliotis larvae. The CRA surfaces and their associated biofilms did not produce GABA from glutamate, and GABA production from putrescine required gabaculine (transaminase inhibitor). The GABA-degrading activity on the CRA surfaces was removed by surface-sterilisation. It ranged from 0.45 to 1.12 nmol cm * h-’ with insignificant seasonal variation. Removal of grazing molluscs from CRA-covered stones led to the growth of a visible biofilm and markedly increased GABA degradation in 2 weeks. Within a few days of placing clean, sterile glass surfaces in the settlement environment, biofilms with GABA-degrading activity developed, and after 11 weeks incubation the GABA degradation rate on glass marbles was 0.37 nmol cmP2 h- ‘. GAB A was not completely oxidised, and the products of GABA metabolism showed similar chromatographic behaviour to alanine and glutamate. In the typical New Zealand Huliotis settlement habitat, GABA
is not likely to play a significant role as an inducer of abalone larval settlement because on the preferred settlement surfaces it is not readily produced from its precursors but it is easily degraded. Keywords: Invertebrate larval settlement; Marine bacteria; Microbial metabolism; Biofilm
1. Introduction Gamma-aminobutyric acid (GABA) inhibits the velar cilia movement in planktonic abalone (Huliotis) larvae, thus promoting their settlement [1,2]. In abalone hatcheries this trait is successfully used to settle competent larvae onto the tank surfaces [3], and a similar role of GABA in the natural Huliotis settlement habitat may be proposed. Marine bacteria
* Corresponding author. Tel: +64-3-548-2319; Fax: +64-3546-9464; Email: [email protected]
can produce GABA from amines or amino acids [4,5], and the ability to degrade GABA is common among marine heterotrophic bacteria [6]. Therefore, marine microorganisms may affect the settlement of Haliotis larvae by affecting the GABA concentration in the settlement environment. The aim of this study was to describe the dynamics of GABA on natural Huliotis settlement surfaces in order to evaluate the role of GABA as a natural settlement inducer. Production and consumption of GABA on the hard surfaces of the coastal marine abalone settlement habitat, particularly on crustose red algae (CRA), are described. GABA was only produced
0168.6496/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SsDl 0168-6496(95)00025-9
206
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
from putrescine in the presence of a transaminase inhibitor, but constitutive GABA-degrading activity in microbial films on settlement surfaces was common. It is unlikely that GABA plays a significant role in Haliatis larval settlement in typical New Zealand Haliotis habitats.
2. Materials
and methods
2.1. GABA analysis GABA was derivatised with phenylisothiocyanate and chromatographed by HPLC [7]. The identity of the GABA peak was established by co-chromatography of the sample mixed with a standard. GABA was measured by peak height comparison with standards. The detection limit for GABA was 0.1 FM. Samples containing radioactive GABA were analysed by the same procedure, except that the injection volume was increased from 4 ~1 to 50 ~1. At an elution rate of 1 ml min-‘, fractions of 0.5 or 1 ml were collected from the detector outlet. These were counted with a Beckman LS 3801 in toluene-based scintillant with Triton-X-100 according to previously published procedures [8]. For the measurement of residual radioactivity, 0.5 ml methanol was added to 0.5 ml sample which was then evaporated to dryness. The residual radioactivity was thus associated with non-volatile compounds. 2.2. Study site and natural GABA concentrations A tidal rock pool in Cable Bay, Nelson, was chosen for the study. The pool was near the low tide level and provided an easily accessible sample of rocky subtidal, the typical habitat of all three New Zealand Haliotis species. The pool contained small stones covered by crustose red algae (mainly Lithothamnion sp.). The solid rock sides were colonised by crustose and articulate corallines (Lithothamnion sp., Corallina sp.) and a variety of foliose seaweeds (Hormosira sp., Carpophyllum sp., Porphyra sp.). Small stones covered by crustose red algae were collected from the tidal rock pool. They were then shaken at 25°C in a small amount of HCl(O.O5-5 N) for l-30 min, to allow for the selective extraction of
Ecology I7 (1995) X-212
amino acids, ranging from those dissolved in the tissue to those bound in peptides. Extracts were analysed for amino acids as above. 2.3. ‘H-GABA -‘H-glutamate
production
from
‘H-putrescine
and
Freshly collected stones (l-2 cm diameter, > 90% surface covered with crustose red algae) were transported in seawater to the laboratory and then placed in duplicate groups of 6-8 in 250-ml beakers, each containing 60 ml of seawater collected from the same area. This water had previously been amended with unlabelled putrescine or glutamate (50-1000 PM) and 1,4 3H-putrescine (0.4 &i ml-‘, specific activity 22.9 Ci mmol- ’ ) or L-[G-3 HIglutamate (0.5 PCi ml-‘, specific activity 50 Ci mmoll ’ 1. Gabaculine (10 pug ml - ‘, inhibitor of GABA transaminase) was added to some beakers, and controls with formalin (0.4% v/v) or without stones were set up. Beakers were incubated at 25°C on a transversal shaker at 120 ‘pm. At various intervals over a 120 h period, 1 ml water samples were removed and stored at - 18°C for later analyses of GABA and radioactivity. At incubation times > 90 h, bacterial growth was observed, and samples were centrifuged at 6000 X g for 15 min at 2°C. Supernatants were stored at - 18°C until they were analysed. 2.4. Measurement
of GABA degradation
Flat, coralline covered stones of l-4 cm diameter were collected from the same rock pool as above. The coralline thalli were polished off the edges of the stones so that the areas of coralline cover could be measured accurately. The stones were separated in four groups, giving total coralline areas of 73-86 cm7 per group. Each group was placed in a 150-ml beaker and covered with seawater (56-69 ml) containing 10 FM GABA. The stones were then incubated at the bulk water temperature in the area where the stones had been collected. Samples (0.5 ml) were taken over a 7-10 h period and analysed for GABA. To determine the effect of season on the GABA degradation rate, the stones were returned to the rock pool immediately after the experiment. They were retrieved at intervals of several weeks during the following year, and the experiment was repeated.
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology Table 1 Seasonal variation of GABA degradation rates on crustose red algal surfaces of small stones in a rock pool at Cable Bay, Nelson Date
7.9.90 3.10.90 16.11.90 3.1.91 27.2.91 15.4.91 12.6.91
Rock pool temperature (“C)
14 16 20 16 27 22 14
a
Experiment temperature (“C)
14 14 19 20 22 22 15
a
Rate +95% conf. interval b (nmol cm-* h-l)
1.10+0.20 0.70 f 0.25 1.07rt 0.07 0.45 rto.04 0.82 F 0.21 1.05rto.19 1.12k0.56
a Depending on the tide and the weather, the water temperaturein the rock pool when the stones were collected was several degrees higher or lower than the subtidal bulk water temperature. The experiments were carried out at the temperature which prevailed in the subtidal on the days before the stones were collected. b Four replicates.
Immediately before these repeats the non-coralline stone surfaces were wiped with acetone. Dates and temperatures for each repeat experiment are given in Table 1. Duplicate control beakers containing no stones, bare stones, or coralline-covered stones which had been wiped with acetone were incubated along with the beakers containing the coralline-covered stones to determine the location and nature of the GABAdegrading activity. To determine the effect of grazing on the rate of GABA degradation, stones (l-2 cm diameter) covered with coralline thalli and various small grazing molluscs were collected from the pool as above. Stones and grazers were brought to the laboratory and kept together in an aquarium until the following day, when the grazers were removed, and the stones divided into 4 groups of 15 stones with similar total surface area. Each group was incubated in 50 ml of seawater containing 50 PM GABA at 23°C on a transversal shaker at 120 ‘pm. Samples (0.1 ml) were taken at intervals of 0.5-l h and analysed for GABA. After 8 h the stones were washed with fresh seawater. Two groups each were placed in 4 1 of aerated seawater supplemented with NaNO, (250 PM), NaSiO, (25 ,uM) and KH,PO, (12 PM). To one aquarium the following grazers were added (numbers in brackets): Diloma nigerrima (4), Melographia aethiops (l), Sypharochiton pelliserpentis (1) and
207
Ecology 17 (199.5) 205-212
Cellana radians (2). The aquaria were exposed to the natural light fluctuations and to temperatures fluctuating between 20 and 24°C. The measurement of GABA degradation was repeated after 7, 15 and 22 days. 2.5. Development
of GABA-degrading
biofilms
Glass marbles (15 mm diameter) were washed with detergent and heat-sterilized. Groups of 16 marbles in plastic netting and individual marbles were placed in a tidal rock pool at Cable Bay. At intervals of several days, marbles were retrieved. Duplicate groups of 8 marbles were incubated in 35 ml seawater containing 10 /.LM GABA at 25°C on a transversal shaker at 120 rpm. Samples (0.5 ml) were taken at intervals and analysed for GABA.
3. Results and discussion 3.1. Natural GABA concentration Extraction of crustose red algae with HCI liberated a complex spectrum of amino acids, but no GABA was found in any sample. The natural GABA pool was therefore below detection limit (0.1 FM or 30 nmol cme2 of crustose red algal surface). This confirms results of other researchers [9]. However, the low GABA concentrations in bulk extracts do not mean that the natural GABA concentration on surfaces is too low to affect abalone larval behaviour. 3.2. Production 3H-glutamate
of 3H-GABA from 3H-putrescine and
GABA has previously been shown to be produced from glutamate and putrescine by isolates of marine bacteria [4,5], but there has been no documentation of the production of the compound at the sites of Huliotis larval settlement. In the absence of gabaculine, incubations of CRA-covered stones with 3Hputrescine and 3H-glutamate (0.05-l mM) resulted in little (< 1 PM), or no GABA production over a 120 h time-course, respectively. In the presence of gabaculine, GABA was produced from labelled putrescine as indicated from the appearance of label (> 90%) in the 6.0 to 6.5 min
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
208 3000
-
A 2500 2000
E.1500
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-
I I
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500
Oh
” yl 9 u)
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U-J
b
d
d
i
d
B
d
d
d
d
Retention
time (min)
I
0
20
40
60
80
100
Time(h)
Fig. 1. A: Profile of 3H-GABA during HPLC of 50 ~1 of sample taken from incubation of 3H-putrescine (1 mM) in the presence of CRA-stones and gabaculine after 96 h. B: Time-course for the production of GABA from 1 mM putrescine in the presence of CRA-stones and gabaculine. Symbols: A, GABA (determined directly by HPLC); v , dpm of GARA per ml of incubation; 0, GARA determined from counts and the specific activity of the starting substrate @Act, 1 X lo6 dpm pmol- ‘).
fraction of a chromatographic run (Fig. lA), corresponding to the retention time of the GABA standard of 6.2 min. No GAEIA was produced from glutamate ( < 0.5 PM). GABA production determined from the counts in the 6.0-6.5 min fractions, and the specific activity of the labelled substrate closely matched that
Ecology I7 (1995) 205-212
determined directly by comparison of peak heights with GABA standard as shown for incubations with 1 mM putrescine (Fig. 1B). The highest GABA concentration resulting from putrescine degradation was 70 PM, which was obtained after 96 h incubation. At starting levels of putrescine of less than 1 mM, maximal levels of GABA did not exceed 45 ,~LMat any stage of the time-course. In controls with stones omitted, or with formalin added, there was no production of GABA. These results indicate GABA as an intermediate, rather than a product of putrescine degradation by microorganisms residing on CFL4-covered surfaces. Although production of GABA from putrescine and glutamate has previously been demonstrated in pure cultures of marine microorganisms [4,5], our experiments showed that GABA accumulated only in the presence of putrescine and the transaminase inhibitor, gabaculine. Several possibilities may explain the failure to detect GABA from glutamate. The first is that label from glutamate would not be incorporated into GABA if it were produced from transamination since glutamate was tritiated only in carbon positions 2 to 4. GABA produced in this way would only utilise the amino group of glutamate, and its formation would be inhibited in the presence of gabaculine. If GABA were produced from decarboxylation of glutamate [lo] then its accumulation would be expected through inhibition of the first step in its utilisation in the presence of gabaculine. This also did not occur. Thus decarboxylation appears unlikely as the means of glutamate conversion to GABA. The conditions under which significant GABA production from putrescine occurred in the presence of the blocking agent, gabaculine, are unlikely to be encountered in the natural coastal environment, and therefore unlikely to operate in the settlement of abalone larvae. It seems more likely that in the natural environment, the presence of the compound is controlled by the enzymes involved in its utilisation. The finding that GABA did not accumulate in the absence of inhibitor suggests that these enzymes effectively remove any GABA that is produced. 3.3. Location, microbial GABA-degrading activity
nature
and products
of
GABA disappeared from water which was incubated in the presence of stones which appeared bare
H.F. Kaspar, D.O. Mountfort/
FEMS Microbiology
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Ecology I7 (1995) 205-212
replicates was often high, so that there was no significant (P = 0.05) seasonal effect on the GABA degradation rate of the CRA surfaces (Table 1). The rock pool temperatures measured when the stones were retrieved ranged from 14°C to 27°C. The actual temperature range was substantially wider due to the extremes of hot summer days and cold winter nights. However, because the pool was submerged for most of the time, the experiments were carried out at the more significant bulk water temperatures (14-22°C).
0
I
’ 0
1
2
3
4
5
<\r 6
7
Time(h)
Fig. 2. Location and nature of GABA-degrading activity. Stones were incubated as described in Materials and methods with GABA at an initial concentration of 10 PM. Symbols: 0, bare stones; +, CRA covered stones; n , water; A, acetone-wiped stones.
or were covered with crustose red algae, but not from water only, or water incubated in the presence of stones which had previously been wiped with acetone (Fig. 2). GABA degradation was therefore carried out by microbes colonising hard surfaces, regardless of whether these surfaces were covered by crustose red algae. Such surfaces are the preferred settlement substrata for abalone larvae [ll-131 and the presence of GABA-degrading activity on these surfaces reduces the likelihood of GABA playing a role in abalone larval settlement. Stones covered in crustose red algae were incubated in seawater containing 10 PM GABA and 0.2 nM 3H-GABA (0.018 &i ml-‘). Samples were taken at intervals for the measurement of GABA, residual radioactivity and radioactive GABA. While GABA and radioactivity in the GABA peak disappeared within a few hours, 63% of the initial 3H appeared in fractions containing glutamate and aspartate. Thus, GABA was largely converted into amino acids which are not inducers of abalone larval settlement ([14], Berkett et al., manuscript in preparation). 3.4. Seasonal [lariation of GABA degrading on crustose red algae
3.5. Effect of grazing on GABA degradation on crustose red algal surfaces
activity
The GABA degradation rate was compared between CRA-covered stones which were either exposed to grazers such as chitons or were free of grazers. During the entire experiment (22 days) the grazed stones remained pink with a clean CRA surface. On the light side of the ungrazed stones a brown diatom film had developed after 7 days. After 15 days small Enteromorpha filaments were visible, and after 22 days these filaments reached 8 mm, and the diatoms had receded. The CRA were covered by a slime. For the first 7 days, GABA degradation was slow, and there was no significant difference in the GABA-degrading activity between grazed and ungrazed stones. Following this initial period, the GABA degradation rate increased on the ungrazed surfaces while it remained low on the grazed surfaces (Fig. 3). Grazing prevented the overgrowing of CRA surfaces by microbial films and thus kept the GABA degrading activity constant and low. The frequent scouring of hard surfaces by grazers appears to have a more significant effect on the GABA-degrading activity than the seasonal temperature changes which are modified by strong tidal temperature fluctuations (Table 1 and Fig. 3). 3.6. Development young biofilms
of GABA-degrading
activity
in
activity
The GABA degradation rates ranged from 0.45 to 1.12 nmol cm-’ h- ‘, but the variability between
GABA-degrading activity can be constitutive in marine bacteria [6]. In order to demonstrate that GABA-degrading activity is constitutive in young marine biofilms, we placed clean, sterile marbles in a
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology
210
tidal rock pool and measured the GABA-degrading activity at intervals of several days. With freshly sterilised marbles, the GABA degradation rate was 30 pmol cme2 h-‘. This activity was assumed to be in the water, and subsequent measurements were corrected for it. Within a few days of placement in the tidal pool, the marbles had a measurable GABA degradation activity (Fig. 4). This activity reached 131 pmol cm-* h-* in 23 days on the marbles which were placed in the pool individually, and 350 pmol cmm2 h-’ i n 77 days on the marbles which were placed in the pool as a group in plastic netting. Probably due to less abrasion, the rate was always higher on the marbles in plastic netting than on the free marbles, many of which were lost so that this
r
::p=
3 $
400
7 _c ys 300 -a $ E p 200 m $ d E 100 5 0 0
IO 20
30
40
50 60
70
80
Time (days)
Fig. 4. Degradation of GABA in young biofilms. GABA was determined in subincubations (1 to 8 h) with marbles taken from tidal rock pool at different intervals over 80 days. Symbols: n , individual marbles; 0, marbles placed in plastic netting.
0 Days
60
Ecology I7 (1995) 205-212
experiment had to be terminated after 23 days. Macro-algal growth and tubeworm deposits developed on the marbles after 20 days and were scraped off before the incubation in presence of GABA, so that the measured rates related to thin microbial biofilms only and were likely to be lower than the total rate. The lower rates on marbles (Fig. 4) compared to CRA-covered surfaces (Table 1) may be a reflection of the pitted CEU surface, where microbes can escape the grazing in conceptacles 19,151.
60 r
3.7. Conclusion
22 Days
0
1
2
3
4
5
6
7
8
Time(h)
Fig. 3. Effect of grazing on GABA-degradation on crustose red algal surfaces. GAE%A was determined in subincubations (1 to 8 h) with stones taken from aquaria at different intervals over 22 days. Symbols: 0, stones exposed to grazers; 0, ungrazed stones (means and standard deviations of duplicates).
Our studies and those of others [9] have failed to demonstrate GABA in coralline algae. The microbial communities on CRA surfaces produced GABA from putrescine only in presence of the GABA transaminase inhibitor gabaculine and did not produce GABA from glutamate. GABA was degraded by the microbial community on the hard surfaces of a typical New Zealand abalone settlement environment. Biofilms with constitutive GABA-degrading activity developed rapidly on clean surfaces. GABA degraders were present on CRA-covered stones yearround and withstood grazing by various molluscs. These results, together with our earlier published data on GABA production and degradation by marine bacteria suggest that GABA is unlikely to play a
H.F. Kaspar, D.O. Mountfort / FEMS Microbiology Ecology 17 (1995) 205-212
significant role as an inducer of abalone larval settlement in a typical New Zealand Haliotis habitat.
Acknowledgements We thank our Cawthron colleagues for valuable discussions. Maggie Atkinson and Jan Holland provided excellent technical assistance. This work was part of contract CAW202 with the New Zealand Foundation for Research, Science and Technology and supported by the New Zealand Lottery Board.
References [l] Morse, D.E. (1992) Molecular mechanisms controlling metamorphosis and recruitment in abalone larvae. In: Abalone of the World: Biology, Fisheries and Culture (Shepherd, S.A., Tegner, M. and Guzman Del Proo, S.A., Eds.), pp. 107-109. Fishing News, Oxford. [2] Morse, D.E., Hooker, N., Duncan, H. and Jensen, L. (1979) Gamma aminobutyric acid a neurotransmitter induces planktonic abalone larvae to settle and begin metamorphosis. Science 204, 407-410. [3] Searcy-Bernal, R., Salas-Garza, A.E., Flores-Aguilar, R.A. and Hinojosa-Rivera, P.R. (1992) Simultaneous comparison of methods for settlement and metamorphosis induction in the red abalone (Haliotis rufescens). Aquaculture 105, 241250. [4] Mountfort, D.O. and Pybus, V. (1992) Effect of pH, temperature and salinity on the production of gamma aminobutyric
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acid (GABA) from amines by marine bacteria. FEMS Microbiol. Ecol. 101, 237-244. [5] Mountfort, D.O. and Pybus, V. (1992) Regulatory influences on the production of gamma-aminobutvric acid by a marine pseudomonad. Appl. Environ. Microbial. 58, 237-242. [61 Kaspar, H.F., Mountfort, D.O. and Pybus, V. (1991) Degradation of gamma aminobutyric acid (GABA) by marine microorganisms. FEMS Microbial. Ecol. 85, 313-318. R.L. and Meredith, S.C. (1984) Amino acid [71 Heinrikson, analysis by reverse-phase high-performance liquid chromatography: precolumn derivatisation with phenylisothiocyanate. Anal. Biochem. 136, 65-74. b31Mountfort, D.O. and Roberton, A.M. (1978) Origin of fermentation products formed during the growth of Bacteroides ruminicola on glucose. J. Gen. Microbial. 106, 353-360. A.L. (1991) [91 Johnson, C.R., Muir, D.G. and Reysenbach, Characteristic bacteria associated with surfaces of coralline algae: a hypothesis for bacterial induction of marine invertebrate larvae. Mar. Ecol. Progr. Ser. 74, 281-294. [lOI Streeter, J.G. and Thompson, J.F. (1972) In vivo and in vitro studies on y-aminobutyric acid metabolism with the radish plant (Raphanus satious L.1. Plant Physiol. 49, 579-584. and [ill Morse, A.N.C. and Morse, D.E. (1984) Recruitment metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae. J. Exp. Mar. Biol. Ecol. 75, 191-215. 1121 Moss, G.A. and Tong, L.J. (1992) Effect of stage of larval development on the settlement of the abalone, Haliotis iris. N.Z. J. Mar. Freshwater Res. 26, 69-73. 1131 Moss, G.A. and Tong, L.J. (1992) Techniques for enhancing larval settlement of the abalone, Haliotis iris, on artificial surfaces. N.Z. J. Mar. Freshwater Res. 26, 75-79. [I41 Morse, D.E., Hooker, N. and Duncan, H. (1980) GABA induces metamorphosis in Haliotis, V: stereochemical specificity. Brain Res. Bull. 5, 381-387. on surfaces of b51 Kaspar, H.F. (1992) Oxygen conditions coralline red algae. Mar. Ecol. Prog. Ser. 81, 97-100.