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Osmoprotection by compatible solutes accumulation in the moderately halophilic bacterium Halomonas elongata DSM 3043
Abstracts / Comparative Biochemistry and Physiology, Part A 126 (2000) S I S163
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C O M P U T E R - A S S I S T E D SPERM ANALYSIS (CASA) AS A TOOL FOR M O N I T O R I N G SPERM QUALITY IN FISH V a n L o o k K.J.W:, MCAllister B . G . , * H u y s k e n s G., * R u r a n g w a E., * O l l e v i e r F. and K i m e D.E. D e p a r t m e n t o f A n i m a l and P l a n t S c i e n c e s , U n i v e r s i t y o f S h e f f i e l d , U . K . and * L a b o r a t o r y f o r A q u a t i c Ecology, Katholieke Universiteit Leuven, Belgium Computer-assisted sperm analysis (CASA) has been increasingly used to examine sperm quality in a range of avian and mammalian species, and more recently has been applied to fish sperm. The Hobson CASA software can continuously track the movement of approximately 200 sperm, producing a quantitative and rapid assessment of sperm motility as shown by 15 different motility parameters including curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), etc. Sperm motility as calculated by CASA is positively related to fertilisation rate and can thus be used to predict the reproductive potential of an individual or extrapolated to populations. The ability of CASA to provide a quantitative and rapid assessment of sperm motility makes it a suitable tool to study the effects of environmental pollutants on sperm quality of both laboratory and wild exposed fish. Mercury decreased the motility of sperm from rainbow trout (Oncorhynchus i~o,kiss), African catfish (Clarias gariepinus), carp (Qvprinus carpio) and goldfish (Carassius auratus) in vitro at concentrations of 0.7, 0.001, 0.1 and 0.1 mg I I, respectively. Tributyltin (TBT) similarly decreased the motility of trout and carp sperm at concentrations of only 0.0001 mg 1 ~ CASA has also shown that sperm motility was decreased in wild roach (Rutilus rutilus) exposed to oestrogenic chemicals. CASA can play an important role in aquaculture as it can rapidly and quantitatively examine the effect of manipulating temperature, photoperiod or holding conditions on sperm quality and hence, fertilising ability of farmed fish. It can be used in assessing the most appropriate extender (diluent) and cryoprotectant to use for any species, the viability of the cryopreserved sperm and the most appropriate temperature for fertilisation. CASA has, for example, been used to select the most suitable cryoprotectant for freezing catfish sperm and assessing the sperm viability after freezing for up to 10 months. Temperature dependence of motility was demonstrated for Oncorhynchus mykiss sperm which was motile for 110 s at 2 °C, but only 65 s at 15 °C, while VCL was similar (around 120 ~m s-r) during the first 15 s after activation at both 2 and 15 °C.
O S M O P R O T E C T I O N BY C O M P A T I B L E SOLUTES A C C U M U L A T I O N IN THE M O D E R A TELY HALOPHILIC B A C T E R I U M H A L O M O N A S E L O N G A TA DSM 3 0 4 3 V a r g a s C., C~inovas D., C a l d e r 6 n M.I., M o r 6 n M.J., C a r r a s c o R., V e n t o s a A. and N i e t o J.J. D e p a r t m e n t o f M i c r o b i o l o g y and P a r a s i t o l o g y , F a c u l t y o f P h a r m a c y , U n i v e r s i t y o f Seville, 4 1 0 1 2 Seville, S p a i n The main strategy used by H. elongata to maintain its internal osmolarity in media with high salinity is the intracellular accumulation of compatible solutes. It is is able of synthesize de novo ectoine and hydroxyectoine when grown in media lacking osmoprotectants and also to accumulate glycine betaine either by direct transport from the external medium or by transport of its precursor, choline, and its subsequent oxidation in two enzymatic steps. Other osmoprotectants such as threhalose, glutamate and glutamine can be synthetized in minor amounts, We have recently isolated and characterized the genetic region involved in ectoine synthesis in this strain, which comprises 3 genes: ectA, which encodes the diaminobutyric acid acetyl transferase, ectB, encoding the diaminobutyric acid aminotransferase, and ectC, encoding the ectoine synthase (2, 3). Although hydroxyectoine can be synthesized directly from ectoine, we have found the existence of an alternative pathway, that converts the precursor NADA into hydroxyectoine without the involvement of ectoine. Besides, we have shown that NADA is an good enzyme stabilizer. Concerning the uptake of osmoprotectants, we have characterized a very efficient system for choline transport and conversion to glycine betaine. The system is mainly regulated by salinity (4, 5). After cloning, sequence analysis of the 4.6 kb region has showed the occurrence of three genes (betlBA), apparently arranged in an operon responsible for the choline-glycine betaine pathway 1. Ventosa A. et al. (1998). Microbiol. Mol. Biol. Rev. 62, 504-544. 2. Cfinovas, D. et al. (1997). J. Biol. Chem.272, 25794-2580 3. C~novas D. et al. (1998). Syst. Appl. Microbiol. 21: 487-497. 4. Cfinovas D. et al. (1996). J. Bacteriol. 178:7221-7226. 5. C~novas, D. et al. (1998). Appl. Environ. Microbiol. 6_44:4095-4097
S 151
C O M P U T E R - A S S I S T E D SPERM ANALYSIS (CASA) AS A TOOL FOR M O N I T O R I N G SPERM QUALITY IN FISH V a n L o o k K.J.W:, MCAllister B . G . , * H u y s k e n s G., * R u r a n g w a E., * O l l e v i e r F. and K i m e D.E. D e p a r t m e n t o f A n i m a l and P l a n t S c i e n c e s , U n i v e r s i t y o f S h e f f i e l d , U . K . and * L a b o r a t o r y f o r A q u a t i c Ecology, Katholieke Universiteit Leuven, Belgium Computer-assisted sperm analysis (CASA) has been increasingly used to examine sperm quality in a range of avian and mammalian species, and more recently has been applied to fish sperm. The Hobson CASA software can continuously track the movement of approximately 200 sperm, producing a quantitative and rapid assessment of sperm motility as shown by 15 different motility parameters including curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), etc. Sperm motility as calculated by CASA is positively related to fertilisation rate and can thus be used to predict the reproductive potential of an individual or extrapolated to populations. The ability of CASA to provide a quantitative and rapid assessment of sperm motility makes it a suitable tool to study the effects of environmental pollutants on sperm quality of both laboratory and wild exposed fish. Mercury decreased the motility of sperm from rainbow trout (Oncorhynchus i~o,kiss), African catfish (Clarias gariepinus), carp (Qvprinus carpio) and goldfish (Carassius auratus) in vitro at concentrations of 0.7, 0.001, 0.1 and 0.1 mg I I, respectively. Tributyltin (TBT) similarly decreased the motility of trout and carp sperm at concentrations of only 0.0001 mg 1 ~ CASA has also shown that sperm motility was decreased in wild roach (Rutilus rutilus) exposed to oestrogenic chemicals. CASA can play an important role in aquaculture as it can rapidly and quantitatively examine the effect of manipulating temperature, photoperiod or holding conditions on sperm quality and hence, fertilising ability of farmed fish. It can be used in assessing the most appropriate extender (diluent) and cryoprotectant to use for any species, the viability of the cryopreserved sperm and the most appropriate temperature for fertilisation. CASA has, for example, been used to select the most suitable cryoprotectant for freezing catfish sperm and assessing the sperm viability after freezing for up to 10 months. Temperature dependence of motility was demonstrated for Oncorhynchus mykiss sperm which was motile for 110 s at 2 °C, but only 65 s at 15 °C, while VCL was similar (around 120 ~m s-r) during the first 15 s after activation at both 2 and 15 °C.
O S M O P R O T E C T I O N BY C O M P A T I B L E SOLUTES A C C U M U L A T I O N IN THE M O D E R A TELY HALOPHILIC B A C T E R I U M H A L O M O N A S E L O N G A TA DSM 3 0 4 3 V a r g a s C., C~inovas D., C a l d e r 6 n M.I., M o r 6 n M.J., C a r r a s c o R., V e n t o s a A. and N i e t o J.J. D e p a r t m e n t o f M i c r o b i o l o g y and P a r a s i t o l o g y , F a c u l t y o f P h a r m a c y , U n i v e r s i t y o f Seville, 4 1 0 1 2 Seville, S p a i n The main strategy used by H. elongata to maintain its internal osmolarity in media with high salinity is the intracellular accumulation of compatible solutes. It is is able of synthesize de novo ectoine and hydroxyectoine when grown in media lacking osmoprotectants and also to accumulate glycine betaine either by direct transport from the external medium or by transport of its precursor, choline, and its subsequent oxidation in two enzymatic steps. Other osmoprotectants such as threhalose, glutamate and glutamine can be synthetized in minor amounts, We have recently isolated and characterized the genetic region involved in ectoine synthesis in this strain, which comprises 3 genes: ectA, which encodes the diaminobutyric acid acetyl transferase, ectB, encoding the diaminobutyric acid aminotransferase, and ectC, encoding the ectoine synthase (2, 3). Although hydroxyectoine can be synthesized directly from ectoine, we have found the existence of an alternative pathway, that converts the precursor NADA into hydroxyectoine without the involvement of ectoine. Besides, we have shown that NADA is an good enzyme stabilizer. Concerning the uptake of osmoprotectants, we have characterized a very efficient system for choline transport and conversion to glycine betaine. The system is mainly regulated by salinity (4, 5). After cloning, sequence analysis of the 4.6 kb region has showed the occurrence of three genes (betlBA), apparently arranged in an operon responsible for the choline-glycine betaine pathway 1. Ventosa A. et al. (1998). Microbiol. Mol. Biol. Rev. 62, 504-544. 2. Cfinovas, D. et al. (1997). J. Biol. Chem.272, 25794-2580 3. C~novas D. et al. (1998). Syst. Appl. Microbiol. 21: 487-497. 4. Cfinovas D. et al. (1996). J. Bacteriol. 178:7221-7226. 5. C~novas, D. et al. (1998). Appl. Environ. Microbiol. 6_44:4095-4097