Some Novel Aerobic Heparin-Degrading Bacterial Isolates

System. Appl. Microbiol. 15, 137-143 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Some Novel Aerobic Heparin-Degrading Bacterial Isolates P. L. STEYN!, B. POT2 , P. SEGERS 2 , K. KERSTERS 2 , and 1

2

3

J. J. JOUBERT3

Dept. of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, Republic of South Africa Laboratorium voor Microbiologie en microbiele Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium Dept. of Medical Microbiology, University of Stellenbosch, 7505 Tygerberg, Republic of South Africa

Received March 15, 1991

Summary Sixteen heparinase producing strains were isolated from soil and activated sludge. Their taxonomic relationships were investigated by polyacrylamide gel electrophoresis of sodium dodecylsulphate denatured whole-cell proteins, by the determination of the guanine plus cytosine content of their genomic deoxyribonucleic acid (DNA) and by DNA: ribosomal ribonucleic acid (RNA) hybridizations with labelled rRNA of Cytophaga heparina LMG 10339T . Three rRNA similarity groups were distinguished. Group I was found to be related to the type strain of Cytophaga heparina and was subdivided in fWO subgroups on the basis of protein electrophoretic profiling and differences in DNA base composition. Group II consisted of only one strain, which showed a lower Tm(e) value with rRNA of C. heparina LMG 10339T and revealed a different electrophoretic protein profile. Group III was found to be quite different from C. heparina. With a T m(e) difference of more than 7°C and a separate position in a numerical analysis of the protein electrophoretic profiles, the latter strains probably constitute a separate genus.

Key words: Heparinase production - Cytophaga heparina - Taxonomy - SDS-PAGE of whole-cell proteins - DNA: rRNA hybridizations Introduction Heparin is a sulphated mucopolysaccharide occurring in liver and lung tissue and also in mast cells. The molecular mass of heparin from different sources varies considerably, from 6.9 X 104 Da (mastocytome tissue of mice; Ogren and Lindahl, 1971) to 1.1 x 10 6 Da (skin of rats, Horner, 1977). The average molecular mass of commercial heparin from porcine intestinal mucosa is 1.3 X 104 Da (Linhardt et al., 1982). The main component of heparin is 2,6-disulphoglucosamine, alternated with uronic acid residues, such as sulphated iduronate and smaller amounts unsulphated glycuronate Uacques and McDuffie, 1978). Heparin is generally used in the prevention and treatment of venous thrombo-embolism. Equipment used for extracorporeal blood circulation, is dependent on heparinization of the patient to prevent blood from clotting in the equipment (Langer et al., 1982 a). The high circulating levels of heparin applied in such cases lead to a high incidence of different complications (Fletcher et al., Abbreviations: SDS-PAGE: Sodium dodecylsulphate polyacrylamide gelelectrophoresis

1976; Gervin, 1975; Langer et al., 1982 a; Swartz and Port, 1979), Langer et al. (1982 b, 1982 c), proposed that a filter with immobilized heparinase be installed between the exit channel of the extra corporeal apparatus and the patient. Heparin can thus be degraded to small polysaccharides without anticlotting properties. Practical application of this filter will lead to an increased demand for heparinase. Heparin degrading enzymes have also found applications in studies on the structure of heparin and related compounds (Dietrich et al., 1973; Linker and Hovingh, 1972; Silva and Dietrich, 1975) and the preparation of heparin fragments as alternative anticoagulant reagents with less side effects (Bergqvist et al., 1985; Diness et al., 1986; Diness and Ostergaard, 1986; Fareed et al., 1988; Linhardt et al., 1982; Matzsch et al., 1987; Neerstand et al., 1987; Ostergaard et al., 1987). An aerobic heparin degrading strain was isolated from soil by Payza and Korn (1956) and described as Flavobacterium heparinum. The description of this species was based on a single isolate. This organism has been transferred to the genus Cytophaga as Cytophaga heparina

138

P. L. Steyn, B. Pot, P. Segers, K. Kersters, and ]. J. Joubert

(Christensen, 1980). Since then, the only other heparin . The remaining 16 strains, together with the authentic C. degrading bacteria reported are species of the anaerobic, heparina strain obtained from the ATee culture collection gram-negative genus Bacteroides [Bacteroides fragilis and an independently received subculture of this strain (Gesner and Jenkin, 1961), Bacteroides thetaiotaomicron (strain AL, Table 1), were investigated by polyacrylamide and Bacteroides ovatus (Salyers et aI., 1977)], Prevotella gel electrophoresis of sodium dodecylsulphate denatured heparinolytica (Gesner and Jenkin, 1961; Shan and Col- whole-cell proteins (SDS-PAGE), by the determination of lins, 1989) and certain species of the gram-positive genera the guanine plus cytosine content of genomic DNA Eubacterium and Peptostreptococcus (Joubert et aI., [%(G+C), in mol%] and by DNA:rRNA hybridizations 1982). Joubert et al. (1984) developed a qualitative in order (i) to group the isolates, (ii) to investigate their method for detecting heparinase producers. Joubert subse- relationship with the only aerobic heparin degrading quently isolated 29 strictly aerobic, gram-negative bacterial isolate known at present (c. heparina), and (iii) heparinolytic bacteria. These isolates were characterized to reveal phylogenetic relationships with reference strains and compared with the authentic C. heparina strain ATee of the genera Bacteroides, Cytophaga, Flavobacterium, 1312ST, but could not be identified (joubert, 1985). Un- Flexibacter, Saprospira and Sphingobacterium. fortunately 13 of these isolates were subsequently lost during storage at -80 o e for 5 years.

Table 1. List of strains investigated Name

LMG number"

Other strain designation b

Strain origin

Cytophaga heparina Cytophaga heparina

10339T,c 10344T,c 10337 10338 10340 10341 10342 10343 10345 10346 10347 10348 10349 9525 10351 9527 10353 10354 8347T 8342T 8340tl d,T 8368 T 1341 T 4008 T 4011 T 10403T 10407T

ATCC 1312ST ALT 113 114 64 66 84 56SY 49 47L 75 10 78 40 63GF 3 lL 43 CCUG 13224T CCUG 11736T CCUG 15907T NCIMB 120S7T ATCC 1341 T ATCC 11947T NCTC 4011 T NCIMB 11299T NCIMB 1363 T NCTC 9343 T ATCC 33268 T

dry soil drysoil Iceland; soil Iceland; soil Brussels, Belgium; soil Riidesheim, Germany; soil Sambyu, Kavango, Namibia; soil Swartkoppies, Transvaal, South Africa; soil Windhoek, Namibia; activated sludge Pretoria, South Africa; soil Western Caprivi, Namibia; soil Pretoria, South Africa; activated sludge Eastern Kavango, Namibia; soil Ephesus, Turkey; soil Amsterdam, the Netherlands; soil Langkloof, Eastern Cape, South Africa; soil Belfast, Eastern Transvaal; soil Roodeplaat, Pretoria, Transvaal, South Africa; soil Uterus Washington, spleen Ventricular fluid of fetus Canada, soil Soil or mud Chalk region, Kent, deep well Human bronchial secretions Soil Woods Hole, Mass, Rockpool near high water Appendix absces Human periodontal pocket

Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase Heparinase

positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive

strain strain strain strain strain strain strain strain strain strain strain strain strain strain strain strain

Sphingobacterium spiritivorum Sphingobacterium spiritivorum Sphingobacterium mizutae Flexibacter canadensis Cytophaga johnsonae Flavobacterium aquatile Flavobacterium breve Flavobacterium ferrugineum Saprospira grandis Bacteroides fragilis e Bacteroides oralis e

T Type strain. " LMG, Laboratorium voor Microbiologie Gent Culture Collection, State University Gent, Belgium. b Strain number as received. Strain numbers of the heparinase positive isolates are as in Joubert, 1985; ATCC, American Type Culture Collection, Rockville, Maryland, U.S.A.; CCUG, Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, University of Goteborg, Sweden; NCIMB, National Collection of Industrial and Marine Bacteria, Torry Research Station, Aberdeen, Scotland; NCfC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. C Strain ATCC B12ST is the type strain of Cytophaga heparina and was received as 'Flavobacterium' heparinum; strain LMG 10344 is an independently received subculture (from Dr. A. Linker, Salt Lake City, Utah, USA) of the type strain of Cytophaga heparina. d t1 indicates that two different colony types were isolated, designated tl and t2. e Lyophilized cells were received from Prof. Dr. W. Mannheim, Zentrum fUr Hygiene und medizinische Mikrobiologie, Klinikum der Philipps-Universitat, D-3550 Marburg-Lahn, Germany.

Novel Heparin-Degrading Bacteria

Materials and Methods Strains used. The bacterial strains examined are listed in Table 1. Maintenance of cultures. Cultures were maintained by weekly transfer on modified Trypticase soy agar [Trypticase soy broth (BBL) with glucose concentration adjusted to 1% and 1.5% Oxoid agar no. 3, w/v] and kept at room temperature. In spite of the fact that these organisms were not fastidious, problems were encountered occasionally with random cultures inexplicably losing viability. Polyacrylamide gel electrophoresis of proteins. All strains were grown on phosphate buffered (0.01 M, pH 7.0) nutrient agar in Roux flasks at 28°C for 32 h. Whole-cell extracts were prepared and SDS-PAGE was performed as described previously (Kiredjian et al., 1986). The normalized densitometric traces of the protein electrophoretic patterns were grouped by numerical analysis using the Pearson product moment correlation coefficient (r) and the techniques as described by Pot et al. (1989). DNA base composition. Cells were grown in Roux flasks on nutrient agar for 2 days. DNA was isolated by the method of Marmur (1961). The average (G+C) content of the DNA, expressed in mol%, was determined by the thermal denaturation method (De Ley and van Muylem, 1963) and calculated using the equation of Marmur and Doty (1962). DNA :rRNA hybridization. 3H-labelled 23S rRNA of C. heparina LMG 10339 T was prepared by using the previously described method (De Ley and De Smedt, 1975). Two mCi[3H]labelled adenine was added to 120 ml modified Trypticase soy broth. The specific activity of the rRNA was 120.103 cpm. ~g-l. Single standed, high molecular weight DNA of the strains from Table 2 was fixed on filters as described by De Ley and De Smedt (1975). DNA: rRNA hybridizations were performed as described by Willems et al. (1989). Each hybrid was characterized by its Tm(e), the temperature in °C at which 50% of the hybrid was denatured.

Results and Discussion Various representatives of the genera Cytophaga, Flavobacterium, and Bacteroides are still poorly classified (Holmes et aI., 1984; Paster et aI., 1985; Reichenbach, 1989; Shah and Collins, 1989, 1990; Woese et aI., 1990). The exact taxonomic position of C. heparina (previously Fl. heparinum), which contains the only aerobic heparinase producing strain known at present, is not completely resolved (Bauwens and De Ley, 1981; Holdeman et aI., 1984; Holmes et aI., 1984; Reichenbach, 1989; Woese et aI., 1990). According to 165 rRNA cataloging, C. heparina belongs to a phylogenetically rather loose major subgroup of the Flavobacterium-Cytophaga group (Paster et aI., 1985). This subgroup contains C. heparina, Flavobacterium ferrugineum, Saprospira grandis, and representatives of the genera Flexibacter and Haliscomenobacter. Another, phylogenetically more tight, subgroup includes the species Flavobacterium aquatile, Flavobacterium uliginosum, Cytophaga johnsonae, Cytophaga lytica and Sporocytophaga myxococcoides. This group also peripherally includes Flavobacterium breve. The DNA: rRNA hybridization technique is a powerful tool to reveal taxonomic relationships at the suprageneric level (De Ley et al., 1978; Pot et al., 1989; Willems et aI.,

139

1989). Extensive studies by DNA: rRNA hybridizations have shown that the Flavobacterium-Cytophaga group belongs to a separate rRNA superfamily (rRNA superfamily V) within the rRNA framework of the gram-negative bacteria (Bauwens and De Ley, 1981). This separate position has been confirmed by the results of Paster et al. (1985).

SDS- PAGE electrophoresis We have shown before, that comparable electrophoresis of whole-cell proteins reveals taxonomic relationships at .the species and subspecies levels (Pot et al. 1989; Vandamme et al., 1990). SDS-PAGE was used here to group the most similar heparinase degrading isolates. The result of a numerical analysis of the SDS-PAGE protein profiles of all strains investigated, using the Pearson product moment correlation coefficient, is presented in Fig. 1. Three main clusters were delineated at the level of r = 0.75. Cluster I is clearly divided in two subclusters. Subcluster I a contains ten isolates, including the two subcultures of the type strain of C. heparina which showed almost identical protein electrophoretic fingerprints. Strains LMG 10342, 9527 and 10354 form a separate protein electrophoretic subcluster Ib (Fig. 1). Cluster II contained only one strain, LMG 10351. This strain differed from the others with respect to (i) the stability of its DNA: rRNA hybrid versus rRNA of C. heparina LMG 10339 T (see below), (ii) its G+C content (41.2 mol %), and (iii) its relative metabolic inertness in various physiological and biochemical tests (Joubert, 1985). Cluster III contained four isolates (Fig. 1). With the exception of strain LMG 10341, all members of cluster III exhibit a hitherto undescribed somersaulting motility. On a microscope slide the cells seem to somersault and then glide from one to two cell lengths. This phenomenon can be observed without any special manipulations. No flagella could be observed in preliminary electron microscope studies.

DNA: rRNA hybridizations DNA: rRNA hybridizations were used to investigate the deeper taxonomic relationships between the different (sub)clusters delineated by SDS-PAGE fingerprinting. Table 2 shows the results of the DNA: rRNA hybridizations performed between labelled rRNA from C. heparina LMG 10339 T and DNAs of strains selected from the results of SDS-PAGE typing. In order to compare our results with the taxonomic scheme presented by Paster et al. (1985), we included also DNAs of the type strains of several members of the C. heparina subgroup of their Flavobacterium-Cytophaga group and DNAs of Cytophaga johnsonae, Fl. aquatile and Fl. breve. We included Flexibacter canadensis, a species incertae sedis of Flexibacter (Reichenbach et al., 1989), which was shown to be a close neighbour of C. heparina (Woese et al., 1990), and representatives of the genus Sphingobacterium (Yabuuchi et al., 1983). From the results presented in Table 2 and Fig. 2, it is obvious that all gram-negative, heparinase producing isolates investigated, belonged to the C. heparina rRNA

140

,

P. L. Steyn, B. Pot, P. Segers, K. Kersters, and]. J. Joubert

0.5

0.7

0.6

0.8

0.9

1.0

Gel electrophoretic protein profile

L..._ _~_ _....L.I_ _--'_ _.....IL.-_----II

LMG no. Cluster no.

9525 10343 10339T 10344T 10349 10347 10346 10353 10348 10345 10342 10354 9527 10351 10337 10340 10341 10338 I 0.5

,

I

I

I

I

0.6

0.7

0.8

0.9

la

LMWM 1.0

Fig. 1. Electrophoretic protein patterns and dendrogram based on unweighted pair group average linkage of correlation coefficients (r) of the protein patterns. Molecular weight markers are indicated by LMWM (from left to right: trypsin inhibitor, 20,100; trypsinogen 24,000; carbonic anhydrase, 29,000; glyceraldehyde-3-phosphate dehydro~enase, 36,000; egg albumin, 45,000; bovine albumin, 66,000; and ~-galactosidase, 116,000). Strain numbers are LMG numbers; indicates type strain.

T m(e)

(OC) 10344T

80

I rRNA

I

subgroup Ib

~

10354

75

-

10343~

10342

r - - : _ - - - - - - - . . , 8527

1034'

8

10351

I~ •• 10341

1033tT

~10348~~~~_ _~~

I rRNA

subgroup Ie

10353

r:lr;:;R~N·A-g-:-r-o-u-p-:I":"1"

337

I rRNA

10338

group III

•

8U7T

•

13UT

•

. . U2T

1340l1T

• 1341T

·4011T

•

10407T

• 4001T

34

10403T •

36

38

40

42

46

48 G.C (mol"') of DNA

Fig. 2. DNA: rRNA similarity map with T m(e) values (in 0c) plotted against the DNA base composition (in mol% G+C). Strain numbers are LMG numbers; T indicates type strain.

Novel Heparin-Degrading Bacteria

branch wit T m(e) values between 80.1 and 70.5°C. They form at least three rRNA groups. For rRNA group I, with Tm(e) values between 80.1 and 76.8 °C, the %(G+C) values clearly show the presence of two subgroups (Table 2, Fig. 1). The rRNA subgroup I a (Tm(e) values between 80.1 and 77.8°C, Table 2) comprises all strains investigated from the protein electrophoretic subcluster I a; rRNA subgroup I b contains the same three isolates of electrophoretic subcluster I b (T m(e) values between 77.4 and 76.8°C, Table 2). The subdivision or rRNA group I was also reflected in the higher (G+C) content of the DNAs of subcluster I a (42.3 to 44.2 mol%, Fig.2), compared with the lower values of subcluster Ib (37.4 to 39.5 mol%, Fig. 2). The (G+C) content of the type strain of C. heparina was found to be 43 mol% [i. e. in the described range of 42 mol% (Bd; as cited by Reichenbach, 1989) to 45 mol% (Tm as

Table 2. Results of DNA:rRNA hybridizations with labeled rRNA of Cytophaga heparina LMG 10339T

determined by Callies and Mannheim, 1980)], wich is in the range measured for representatives of rRNA subgroup I a. The rRNA group II contains only one strain, LMG 10351 (Tm(e) value of 74.2°C, (G+C) content 41 mol%, Table 2 and Fig. 2). The separate position of this strain on the C. heparina rRNA branch is in agreement with its protein electrophoretic position (Fig. 1). In rRNA group III we found three strains with T m(e) values between 70.9 and 70.5°C and (G+C) content of about 37 mol%. These three strains were all representatives of the protein electrophoretic cluster III. The rRNA group III is quite distinct from rRNA groups I and II according to (i) the stability of their DNA: rRNA hybrids versus rRNA of C. heparina LMG 10339T , (ii) their electrophoretic protein patterns, and (iii) their morphological properties as found by Joubert (1985). This group of

Strain number"

Name

rRNA group I rRNA subgroup Ia

Cytophaga heparina Cytophaga heparina

Heparinase Heparinase Heparinase Heparinase Heparinase

positive positive positive positive positive

strain strain strain strain strain

LMG LMG LMG LMG LMG LMG LMG

10339T 10344T 9525 10343 10348 10349 10353

G+C content (mol%)

T m(e).in DC with 3H-labeled 23S rRNA of C. heparina LMG 10339T

43.0 42.3 42.9 42.3 44.1 43.7 44.2

80.0 80.1 79.3 78.9 78.2 77.9 77.8

rRNA subgroup Ib Heparinase positive strain Heparinase positive strain Heparinase positive strain

LMG 10342 LMG 9527 LMG 10354

39.5 37.5 37.4

77.4 77.0 76.8

rRNA group II Heparinase positive strain

LMG 10351

41.2

74.2

rRNA group III Heparinase positive strain Heparinase positive strain Heparinase positive strain

LMG 10337 LMG 10341 LMG 10338

37.0 37.1 36.9

70.9 70.6 70.5

LMG 8347T LMG 8342 T LMG 8340t1 T LMG 8368 T LMG 1341 T LMG 4008 T LMG 4011 T LMG 10403 T LMG 10407T NCTC 9343 T ATCC 33269T

39.8 40.5 39.3 38.4 36.2 34.1 34.7 48.9 46.3 53.5 41.1 b

69.5 67.4 67.3 67.9 58.5 55.7 58.3 55.7 57.1 52.9 51.8

Reference organisms

Sphingobacterium spiritivorum Sphingobacterium multivorum Shingobacterium mizutae Flexibacter canadensis Cytophaga johnsonae Flavobacterium aquatile Flavobacterium breve Flavobacterium ferrugineum Saprospira grandis Bacteroides fragilis Bacteroides oralis

t

a

b

141

Type strain; t1 indicates that two different colony types were isolated, designated t1 and t2. LMG, Laboratorium voor Microbiologie Gent Culture Collection, State University Gent, Belgium; ATCC, American Type Culture Collection, Rockville, Maryland, U.S.A.; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. According to Watabe et aI., 1983.

142

P.L.Steyn, B.Pot, P.Segers, K.Kersters, andJ.J.Joubert

isolates probably represents a separate taxon with the rank of genus. Further investigations, including DNA: rRNA hybridizations, are presently being performed to investigate the exact taxonomic status of these organisms. From the reference strains included, Sphingobacterium

spiritivorum, Sphingobacterium multivorum, Sphingobacterium mizutae and Flex. canadensis displayed the highest Tm(e) values versus rRNA of C. heparina LMG 10339 T (Table 2, Fig. 2). The Sphingobacterium species were first described as members of Flavobacterium but were separated on the basis of fatty acid composition and the typical presence of sphingolipids (Yabuuchi et ai., 1983; Dees et ai., 1985; Holmes et ai., 1988). Our DNA: rRNA hybridization results support the separate position of Sphingobacterium in the Flavobacterium-Cytophaga complex (Table2, Fig. 2). No significant relationship was found by DNA:rRNA hybridizations with any of the other reference strains of Flavobacterium, Cytophaga or Bacteroides. This is in good agreement with the earlier results of Bauwens and De Ley (1981), Paster et ai. (1985), and Woese et ai. (1990). Our results indicate that heparinase production by aerobic gram negative bacteria is not confined to a single taxon. The new heparinase positive isolates display considerable genotypic and phenotypic heterogeneity, although most of them are located on the C. heparina rRNA branch. The exact degree of relatedness between the different heparinase producing bacteria and other possibly related organisms of the genera Cytophaga, Flavobacterium, Flexibacter and Sphingobacterium should be further investigated by a polyphasic taxonomic approach, including DNA: DNA hybridizations.

Acknowledgements. We thank D. Dewettinck for excellent technical assistance. Part of this research was carried out in the framework of contract BAP-0138-B of the Biotechnology Action Program of the Commission of the European Communities. K. Kersters is indebted to the 'Fund for Medical Scientific Research', Belgium, for research and personnel grants. The University of Pretoria is thanked for partially sponsoring P. L. Steyn. References

Bauwens, M., De Ley, j.: Improvements in the taxonomy of Flavobacterium by DNA: rRNA hybridizations. pp. 27-31. In: The Flavobacterium-Cytophaga group. Proceedings of the international symposium on yellow-pigmented gram-negative bacteria of the Flavobacterium-Cytophaga group (H. Reichenbach, and O.B. Weeks, eds.). Weinheim, Verlag Chemie 1981 Bergqvist, D., Nilsson, B., Hedner, U., Pedersen, P. c., Ostergaard, P. B.: The effect of heparin fragments of different molecular weights on experimental thrombosis and haemostasis. Thromb. Res. 38, 589-601 (1985) Callies, E., Mannheim, W.: Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains. Antonie V. Leeuwenhoek 46,41-49 (1980) Christensen, P.: Description and taxonomic status of Cytophaga heparina (Payza and Korn) comb. nov. (Basionym: Flavobacterium heparinum Payza and Korn 1956). Int. J. System. Bact. 30,473-475 (1980)

Dees, S. B., Carlone, G. M., Hollis, D., Moss, C. W.: Chemical and phenotypic characteristics of Flavobacterium thalpophilum compared with those of other Flavobacterium and Sphingobacterium species. Int. J. System. Bact. 35, 16-22 (1985) De Ley, j., De Smedt, j.: Improvements of the membrane filter method for DNA:rRNA hybridization. Antonie V. Leeuwenhoek 41,287-307 (1975) De Ley, j., van Muylem, j.: Some applications of deoxyribonucleic acid base composition in bacterial taxonomy. Antonie V. Leeuwenhoek 29,344-358 (1983) De Ley, j., Segers, P., Gillis, M.: Intra- and intergeneric similarities of Chromobacterium and janthinobacterium ribosomal ribonucleic acid cistrons. Int. J. System. Bact. 28, 154-168 (1978) Dietrich, C. P., Silva, M. E., Michelacci, Y. M.: Sequential degradation of heparin in Flavobacterium heparinum. Purification and properties of live enzymes involved in heparin degradation. J. BioI. Chern. 248, 6408-6415 (1973) Diness, V., Nielson, j. r., Pedersen, P. c., Wolffbrandt, K. H., Ostergaard, P. B.: A comparison of the antithrombotic and haemorrhagic effects of a low molecular weight heparin (LHN-1) and conventional heparin. Thromb. Res. 55, 410-414 (1986) Diness, V., Ostergaard, P. B.: Neutralization of a low molecular weight heparin (LHN-1) and conventional heparin by protamine sulfate in rats. Thromb. Haemostasis 56, 318-322 (1986) Fareed, j., Walenga, j. M., Hoppenstead, D., Huan, X., Racanelli, A.: Comparative study on the in vitro activities of seven low molecular weight heparins. Haemostasis 18, 3-15 (1988) Fletcher, j. R., McGee, A. E., Mills, M., Snyder, K. c., Herman, C. M.: Twenty four hour membrane oxygenation in dogs without anticoagulation. Surgery 80, 214-223 (1976) Gervin, A. S.: Complications of heparin therapy. Surg. Gynee. Obstet. 140, 789-796 (1975) Gesner, B. M., jenkin, C. R.: Production of heparinase by Bacteroides. J. Bact. 81, 595-604 (1961) Holdeman, L. V., Kelley, R. W., Moore, W. E. c.: Genus I. Bacteroides Castellani and Chalmers 1919, 959 AL , pp.604-631. In: N. R. Krieg and j. G. Holt (eds.), Bergey's manual of systematic bacteriology, vol. 1. Baltimore, The Williams & Wilkins Co. 1984 Holmes, B., Owen, R. J., McMeekin, T. A.: Genus Flavobacterium Bergey, Harrison, Breed, Hammer and Huntoon 1923, 97 AL , pp.353-361. In: N. R. Krieg and j. G. Holt (eds.), Bergey's manual of systematic bacteriology, vol. 1. Baltimore, The Williams & Wilkins Co. 1984 Holmes, B., Weaver, R. E., Steigerwalt, A. G., Brenner, D. j.: A taxonomic study of Flavobacterium spiritivorum and Sphingobacterium mizutae: proposal of Flavobacterium yabuuchiae sp. nov. and Flavobacterium mizutaii comb. nov .. Int. J. System. Bact. 38, 348-353 (1988) Horner, A. A.: The concept of macromolecular heparin and its physiological significance. Fed. Proe. 36, 35-39 (1977) jacques, L. B., McDuffie, N. M.: Heparin: The chemical and anticoagulant nature of heparin. Seminars Thromb. Hemost. 4, 277-297 (1978) joubert, j. j.: Kenmerking van heparinase produserende mikroorganismes. M. D. thesis, University of Pretoria, Pretoria, RSA (1985) joubert, j. j., Greer, A. S., du Preez, j. H.: Heparin degradation by Eubacterium and Peptostreptococcus species from bovine' endometritis. J. S. Afr. Vet. Ass. 53, 214 (1982) Joubert, j. j., Jansen van Rensburg, E., Pitout, M. j.: A plate method for demonstrating the breakdown of heparin and

Novel Heparin-Degrading Bacteria chondroitin sulphate by bacteria. MicrobioL Meth. 2, 197-202 (1984) Kiredjian, M. , Holmes, B., Kersters, K., Guilvout, I., De Ley, j.: Alcaligenis piechaudii, a new species from human clinical specimens and the environment. Int. J. System. Bact. 36, 282-287 (1986)

Langer, R., Linhardt, R. J., Cooney, C. 1., Tapper, D., Klein, M.

D.: Immobilized heparinase: Production, purification and applications in extra corporeal therapy. Enzyme Engl. 6,433-441 (1982 a) Langer, R., Linhardt, R. j., Galliher, P. M., Flanagan, M. M., Cooney, C. 1., Klein, M. D.: A system for heparin removal. pp.493-509. In: Biomaterials: Interfacial phenomena and application (S. Cooper, A. Hoffman, B. Ratner, N. A. Peppas eds.). Am. Chern. Soc. 1982 b Langer, R., Linhardt, R. j., Hoffberg, S., Larsen, A. K., Cooney, C. L., Tapper, D., Klein, M.: An enzymatic system for removing heparin in extracorporeal therapy. Science 217, 261-263 (1982c) Linhardt, R. j., Grant, A., Cooney, C. L., Langer, R.: Differential coagulant activity of heparin fragments prepared using microbial heparinase. J. BioI. Chern. 257, 7310-7313 (1982) Linker, A., Hovingh, P.: Isolation and characterization of oligosaccharides obtained from heparin by action of heparinase. Biochemistry 11, 563-568 (1972) Mdtzsch, T., Bergqvist, D., Hedner, V., Ostergaard, P. B.: Effects of an enzymatically depolymerized heparin as compared with conventional heparin in healthy volunteers. Thromb. Haemostasis 57, 97-101 (1987) Marmur, j. A. : A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. BioI. 3, 208-218 (1961) Marmur, j. A., Doty, P.: Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. BioI. 5, 109-118 (1962) Neerstand, H., Ostergaard, P. B., Bergqvist, D., Mdtsch, T., Hedner, V.: tPA inhibitor, tPA :Ag, plasminogen and 2-antiplasmin after low molecular weight heparin or standard heparin. Fibrinolysis 1, 39-43 (1987) Ogren, S., Lindahl, V.: Degradation of heparin in mouse mastocytoma tissue. Biochem. J. 125, 1119-1129 (1971)

Ostergaard, P. B., Nilsson, B., Bergqvist, D., Hedner, V., Pedersen, P. c.: The effect of low molecular weight heparin on

experimental thrombosis and haemostasis - the influence of production method. Thromb. Res. 45, 739-749 (1987) Paster, B. j., Ludwig, W., Weisburg, W. G., Stackebrandt, E., Hespell, R. B., Hahn, C. M., Reichenbach, H., Stetter, K. 0 ., Woese, C. R.: A phylogenetic grouping of the bacteroides, cytophagas and certain f1avobacteria. System. Appl. Microbiol. 6, 34-42 (1985) Payza, A. N., Korn, E. D.: The degradation of heparin by bacterial enzymes. I. Adaptation and lyophilized cells. J. BioI. Chern. 223, 853-864 (1956)

Pot, B., Gillis, M., Hoste, B., Van de Velde, A., Bekaert, F., Kersters, K., De Ley, j. : Intra- and intergeneric relationships of

143

the genus Oceanospirillum. Int. J. System. Bact. 39, 23-34 (1989) Reichenbach, H.: Genus!. Cytophaga Winogradsky 1929, 577,AL emend., pp. 2015-2050. In: T. Staley, M. P. Bryant, H. Pfennig and j. G. Holt (eds.), Bergey's manual of systematic bacteriology, vo!' 3. Baltimore, The Williams & Wilkins Co. 1989 Salyers, A. A., Vercelotti, J. R., West, S. E. H, Wilkens, T. D.: Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl. Environ. MicrobioL 33, 319-322 (1977) Shah, H N., Collins, M. D.: Proposal to restrict the genus Bacteroides (Castellani and Chalmers) to Bacteroides fragilis and closely related species. Int. ]. System. Bact. 39, 85-87 (1989) Shah, H N., Col/ins, M. D.: Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int. J. System. Bact. 40, 205-208 (1990) Silva, M. E., Dietrich, C. P.: Structure of heparin: characterization of the products formed from heparin by action of heparinase and heparitinase from Flavobacterium heparinum. J. BioI. Chern. 250, 6841-6848 (1975) Swartz, R. D., Port, F. K.: Preventing hemorrhage in high risk hemodialysis: Regional versus low-dose heparin. Kidney Int. 16,513-518 (1979) Vandamme, P., Pot, B., Falsen, E., Kersters, K., De Ley, j.: Intraand interspecific relationships of veterinary campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA: DNA hybridizations. System. App!. Microbio!. 13, 295-303 (1990) Watabe, j., Benno, Y. , Mitsuoka, j.: Taxonomic study of Bacteroides oralis and related organisms and proposal of Bacteroides veronalis sp. nov .. Int. J. System. Bact. 33, 57-64 (1983) Willems, A., Busse, j., Goor, M., Pot, B., Falsen, E., Jantzen, E.,

Hoste, B., Gillis, M., Kersters, K., Auling, G., De Ley, j.: Hydrogenaphaga, a new genus of hydrogen-oxidizing bacteria that includes Hydrogenophaga (lava comb. nov. (formerly Pseudomonas (lava), Hydrogenophaga palleroni, (formerly Pseudomonas palleroni), Hydrogenophaga pseudo(lava (formerly Pseudomonas pseudo(lava and Pseudomonas carboxydoflava) , and Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis). Int. J. System. Bact. 39, 319-333 (1989)

Woese, C. R., Yang, D., Mandelco, 1., Stetter, K. 0.: The Flexibacter-Flavobacter connection. System. App!. Microbiol. 13, 161-165 (1990)

Yabuuchi, E., Kaneko, T., Yano, I., Moss, C. W., Miyoshi, N.: Sphingobacterium gen. nov., Sphingobacterium spiritivorum comb. nov., Sphingobacterium multivorum comb. nov., Sphingobacterium mizutae sp. nov., and Flavobacterium indologenes sp. nov.: Glucose-nonfermenting gram-negative rods in CDC Groups IIK-2 and II b. Int. (1983)

J. System.

Bact. 33, 580-598

Professor Dr. P. 1. Steyn, Dept. of Microbiology and Plant Pathology, University of Pretoria, 0002 Pretoria, Republic of South Africa