Polyphosphate-dependent enzymes in some coryneform bacteria isolated from sewage sludge

FEMS MicrobiologyLetters 107 (1993) 133-138 © 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00 Published by Elsevier 133 FEMSLE 05280 Kerstin Bark, Peter Kiimpfer, Alexander Sponner and Wolfgang Dott Fachgebiet Hygiene, Technische Universitiit Berlin, Berlin, FRG (Received 18 August 1992; revision received 24 November 1992; accepted 30 November 1992) Abstract: Eleven isolates obtained from a laboratory sewage treatment plant, most of them presumptively assigned to the coryneform genera Curtobacterium and Aureobacterium were studied for the presence of intracellular polyphosphates and polyphosphate dependent enzymes. All isolates stored polyphosphates and showed adenylate kinase activities ranging from 64 to 815 mU mg -1. Polyphosphate:AMP phosphotransferase could only be detected in one isolate. Three isolates showed a polyphosphate kinase activity also in minor amounts from 15 to 17 mU mg -1. A polyphosphate dependent NAD or 3-phosphoglycerate kinase could not be detected. Polyphosphate glucokinase activity was measured in cell-free extracts of nine isolates ranging from 2 to 376 mU mg-1. Three isolates showed in addition to the polyphosphate glucokinase, a glucose-6-phosphate-dependent NAD kinase. For the regeneration of NADP from NAD and polyphosphate, this enzyme system may give the isolates a distinct competitive advantage, especially for anabolic processes. The polyphosphate-dependent enzymes reported here may play an additional role in the complex process of 'biological' phosphate removal from wastewater. Key words: Polyphosphate accumulation; 'Biological' phosphate removal; Coryneform bacteria; Polyphosphate glucokinase; Glucose-6-phosphate-dependent NAD kinase Introduction Excess ' b i o l o g i c a l ' p h o s p h a t e r e m o v a l in sewage t r e a t m e n t plants is generally a t t r i b u t e d to the a c c u m u l a t i o n of i n t r a c e l l u l a r p o l y p h o s p h a t e by bacteria, which can act as reserve m a t e r i a l or might be u s e d as e n e r g y reserve [22,24]. F o r the complex process, several models have b e e n proposed [15,24], which all d e p e n d heavily o n the p r e d o m i n a n c e of Acinetobacter spp. or r e l a t e d Correspondence to: P. K~mpfer, Fachgebiet Hygiene, Technische Universit~it Berlin, Amrumerstr. 32, D-1000 Berlin 65, FRG. organisms in sewage p l a n t s [24]. A l t h o u g h the enzymes polyphosphate : AMP phosphotransferase a n d a d e n y l a t e kinase have b e e n shown to form A T P from native a n d synthetic polyphosp h a t e in strain Acinetobacterjohnsonii 210A [5,22] a n d several o t h e r strains b e l o n g i n g to the g e n u s Acinetobacter [12], the role of this e n e r g y g e n e r a t ing process within the overall process of 'biological' p h o s p h a t e removal is far from clear. Several reports described the p r e d o m i n a n c e of o t h e r genera t h a n Acinetobacter in activated sludges showing e n h a n c e d p h o s p h a t e removal [3,13,19,21]. I n the p r e s e n t study it is shown that some c o r y n e f o r m b a c t e r i a isolated from a l a b o r a t o r y Polyphosphate-dependent enzymes in some coryneform bacteria isolated from sewage sludge 134 pilot plant showing enhanced phosphate removal, possessed a polyphosphate glucokinase and in Biochemical properties and phenotypic characterization some cases a glucose-6-phosphate-dependent NAD kinase. The possible role of these enzymes in t h e o v e r a l l p r o c e s s o f ' b i o l o g i c a l ' p h o s p h a t e r e m o v a l is d i s c u s s e d . After study of cell morphology (Leitz, Wetzlar, FRG), Gram-staining (modified Hucker method), o x i d a s e t e s t i n g ( M e r c k , B a c t i d e n t 13000, D a r m - database using the methods of numerical c a t i o n a s d e s c r i b e d p r e v i o u s l y [14]. Materials and Methods identifi- Organisms and growth conditions T h e 11 o r g a n i s m s i n c l u d e d i n t h i s s t u d y ( T a b l e 1) w e r e i s o l a t e d f r o m a l a b o r a t o r y p i l o t p l a n t showing enhanced phosphate removal. They were cultivated aerobically on nutrient agar (Difco) at Cellular fatty acids Cellular fatty acids were transmethylated with methanolic HCI, and fatty acid methyl esters were analysed by gas liquid chromatography using a Shimadzu GC 9A equipped with a fused silica c a p i l l a r y c o l u m n (50 m × 0 . 3 2 m m ) a s r e p o r t e d e l s e w h e r e [12]. 25°C. For studying enzyme activities, cells were grown in a modified medium previously reported [11] w i t h 5 0 0 m g 1-1 y e a s t e x t r a c t . Table 1 Specific activities of different polyphosphate-dependent enzymes and presence of polyphosphate fractions according to Clark et al. [7] in cell-free extracts of eleven isolates Isolate No. 7 8 2 103 24 39 101 136 27 14 109 a b c d e Identification result a Enzyme activities d,e PPT AK PK PG GNK PNK PGK Aureobacterium saperdae Aureobacterium saperdae Cellulomonas cellulans-like Cellulomonas cellulans-like Curtobacterium sp. Curtobacteriurn sp. Curtobacteriurn sp. Curtobacterium sp. 10 144 177 250 293 133 362 219 64 309 251 815 17 16 15 . - 262 48 6 33 254 293 376 82 84 - - - - - Not identified b Shewanella putrefaciens like Not identified c - . . 6 76 . 21 18 . . . . PolyP content f'g II III + _+ 5: + 5: 5: 5: 5: + 5: + + + + + + + + + + 5: + Identification results are based on phenotypic characterization and cellular fatty acid profiles, for details see text. Gram-positive, oxidase-negative irregular rod. Gram-negative, oxidase-positive rod with large amounts of intracellular granules. Enzyme activities are given as specific activity in m U / m g protein (nmol substrate per min and mg protein). Enzyme abbreviations: PPT, polyphosphate:AMP phosphotransferase; AK, adenylate kinase; PK, polyphosphate kinase; PG, polyphosphate glucokinase; GNK, glucose-6-phosphate-dependent NAD kinase; PNK, polyphosphate-dependent NAD kinase; PGK, polyphosphate-dependent 3-phosphoglycerate kinase. f Intensities of PAGE bands after staining with Toluidine blue: + + , moderate intensity; + , low intensity; - , no band present (for comparison see reference 4). g For isolates Nos. 2, 7, and 39, the total P content ( m g P / g dry weight) was 49, 38, and 73, respectively. s t a d t , F R G ) , all i s o l a t e s w e r e c h a r a c t e r i z e d p h e n o t y p i c a l l y w i t h 87 p h y s i o l o g i c a l t e s t s a n d t h e resulting test profiles were compared with a 135 Electron microscopy Extraction and PAGE of polyphosphates Extraction of polyphosphate was performed according to Clark et al. [6] with minor modifications (temperature 4°C; incubation times 10-20 rain between the steps). Polyacrylamide gel electrophoresis (PAGE) was performed with Trisborate buffer, pH 8,3 for 10% gels and ran at 200 V for 1 h using a Mini Protean 2 Cell (BioRad Laboratories, Munich, FR (...truncated)