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Tyvelose in Agromyces Cell Walls
System. Appl. Microbiol. 15, 187-190 (1992) © Gustav Fischer Verlag, StuttgartlNew York
Tyvelose in Agromyces Cell Walls 1. 1. MALTSEV1, A. I. KALINOVSKyl, HELEN I. ZGURSKAYA2 , and LYUDMILA I. EVTUSHENK0 2 * 1 2
Pacific Institute of Bioorganic Chemistry, Academy of Sciences, Vladivostok 690 022, Russia Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences, Pushchino, Moscow Region 142 292, Russia
Revision received December 6, 1991
Summary Tyvelose (3,6-dideoxy-D-mannose or 3,6-dideoxy-D-arabinohexose) was found in hydrolysates of whole cells and native cell walls of Agromyces cerinus ssp. cerinus and some whole cell samples of Agromyces ramosus type strain (trace). The sugar was isolated, purified and identified by thin-layer and gas liquid chromatography, mass spectrometry, proton eH) and BC nuclear magnetic resonance (NMR) spectroscopy as well as by optical spectrophotometry . Tyvelose was not found in the cell wall of other actinomycetes and Gram-positive bacteria tested so far.
Key words: Agromyces - Clavibacter - 3,6-Dideoxy-D-arabinohexose - 3,6-Dideoxy-D-mannose - Tyvelose - Cell wall Introduction We recently detected in whole cell hydrolysates of the aerobic actinomycete Agromyces cerinus ssp. cerinus (Evtushenko et ai., 1991; Zgurskaya et ai., 1991) a hexose constituent that was not found previously in actinomycetes and other Gram-positive bacteria. This paper presents its identification as 3,6-dideoxy-D-arabinohexose (or 3,6-dideoxy-D-mannose, or tyvelose). Materials and Methods Bacterial strains and culture conditions. We used fifteen strains
of Agromyces, A. ramosus ATCC 17325 (Casida, 1986) and A.
cerinus ssp. cerinus VKM Ac-1340T, VKM Ac-1341, VKM Ac-1342, VKM Ac-1343, VKM Ac-1344, VKM Ac-1350, A. cerinus ssp. nitratus VKM Ac-1351 T, A. (ucosus ssp. (ucosus VKM Ac-1345 T, VKM Ac- 1346, VKM Ac-1347, VKM Ac-1348, VKM Ac-1349, A. (ucosus ssp. hyppuratus VKM Ac-1352T, VKM Ac-1353 (Zgurskaya et aI., 1991) together with 46 representatives of the genus Clavibacter (Davis et aI., 1984), including the type strains C. michiganensis ssp. michiganensis ICMP 2550 T (= NCPPB 2979 T), C. michiganensis ssp. insidiosus ICMP 2621 T (= NCPPB 1109 T), C. michiganensis ssp. nebraskensis ICMP 3298 T (= NCPPB 2581 T ), C. michiganensis ssp. sepedonicus ICMP 2535 T (= NCPPB 2137T) C. michiganensis ssp. tesselarius ICMP 7221T (= ATCC 33566T), C. iranicus
* Corresponding author
ICMP 3496T (= NCPPB 2253 T), C. rathayi ICMP 2574 T (= NCPPB 2980T), C. tritici ICMP 2626T (= ATCC 11403 T ), obtained from Dr. J. M. Young (Auckland, New Zealand). VKM, All-Union Collection of Microorganisms, Institute of biochemistry and physiology of microorganisms, Puschino, Moskow region, USSR; ICMP, International collection of microorganism from plants, Plant diseases division, DSIR, Auckland, New Zealand; NCPPB, National collection of Plant Pathogenic bacteria, Plantpathology laboratory, Hapenden, Hertsfordshire, U.K. The cultures were grown aerobically in a liquid medium commonly used for phytopathogenic corynebacteria (casein peptone, 10 g; yeast extract,S g; glucose,S g; NaCl, 5 g; distilled water, 1 I) at 30°C for 24 h as well as on solid mineral medium (Pridham and Gottlieb, 1948) with glucose, rhamnose, mannose, galactose, or xylose as carbon source and addition of yeast extract and casamino acids (by 0.1%) containing agar (1,5%). Preparation o( cells and cell walls. The cultures were washed with saline solution (0.8% NaCl) and a mixture of chloroform and methanol (1 : 1, v/v) and air dried. To obtain cell walls, living cells were disrupted using a ball mill with glass beads; the cell \\";llls were separated by differential centrifugation and repeatedly wah sed with water and a chloroform-methanol (1: 1, v/v) mixture and air dried. Chemical analysis o( sugars. The sugar composition of various acid hydrolysates (hydrolysis conditions: 0.5 M HCl, 120 °C, 30 min; 1 M trifluoroacetic acid, 100°C, 1 h; 2 M H 2S0 4 , 100°C, 1 h) was assayed according to the earlier described chromatography methods (Hasegawa et aI., 1983; Lechevalier and Lechevalier, 1980) using Cellulose (Merck, Art. 5716) and
188
1.1. Maltsev, A.1. Kalinovsky, H.1. Zgurskaya, and L.I. Evtushenko
Kieselgel 60 (Merck, Art. 5719) plates as well as by gas liquid chromatography. Acetylated aldonitriles were analyzed by gas chromatography (Easterwood and Huff, 1969). A Tsvet chromatograph (USSR) was used: glass column (2 m X 3 mm LD.)j packed phase, 3% QF on 100/120 mesh Cromatron NAWHMDSj carrier gas argon (60 cm 3/min); the temperature program was 150 0-100/min-220 °C. To identify the unknown sugar, an LKB-9000 gas chromatograph-mass spectrometer was employed with the following characteristics: column (3 m X 3 mm) with 0.1 % QF-1 on a Chromatron NAW-HMDSj carrier gas helium (50 cm 3/min), column temperature program 150o-10olmin-220°Cj ionization voltage, 70 eV; temperature of the ionic source, molecular sefarator and evaporator, 225, 265 and 275°C, respectively. The Hand 13C NMR spectra were recorded by a Bruker WH-250 spectrometer in D2 0 . Optical rotation was measured using a Model 141 Perkin Elmer polarimeter by D-line irradiation in the sodium lamp spectrum.
Isolation and purification of the unknown sugar Dry biomass (5 g) of strain A. cerinus ssp. cerinus VKM Ac-1340 was heated for 1.5 h in 20 ml of 1 M trifluoroacetic acid at 100°C and centrifuged. The pellet was discarded, the supernatant was evaporated and dried under vacuum. Nearly 2 g of the darkbrown precipitate was formed and then dissolved in 5 ml of water with subsequent addition of 60 ml ethanol. The precipitate formed was separated again by centrifugation. After evaporation of the supernatant under vacuum the dry pellet (1 g) was applied to a column with L 40/100 silica gel in the solvent system chloroform:methanol:water (75: 25 : 1, vi v). The separation was controlled by thin-layer chromatography on silica gel plates in the same solvent system. The fractions containing the chromatographically pure monosaccharide were combined and evaporated under vacuum. White powder (22 mg) was obtained.
Results and Discussion
Identification of the unknown sugar
Acid hydrolysis of the preparations and the detection of the unknown sugar. The analysis of various acid hydrolysates of whole cell and native cell wall preparations in some solvent systems revealed a sugar with an Rf exceeding that of rhamnose and differing by its properties from other highly mobile sugars previously found in aerobic actinomycetes (Asano et aI., 1989; Hasegawa et aI., 1983; Lechevalier and Lechevalier, 1986) (Table 1). The sugar gave a brown colour with aniline phthalate. The sugar could not be detected when H 2 S0 4 was used for hydrolysis. (In the method of Lechevalier and Lechevalier (1980) which is often employed for whole cell hydrolysates of actinomycetes H 2 S0 4 is used as acid). The analysis of the sugar acetylated aldonitriles by gas liquid chromatography also revealed a peak with a retention time shorter than that of the rhamnose derivative and differing from those derivatites of other sugars of the standard solution (glucose, galactose, arabinose, mannose, ribose and xylose).
The mass spectrum of the compound obtained by gas liquid chromatography and mass spectrometry was identical to that of paracetate aldonitrile 3,6-dideoxy-hexose (m/z: 212, 184 (100), 142, 129, 128) (Atlas of the mass spectra of organic compounds, 1984). To determine the configuration of its asymmetric centres, the monosaccharide was isolated individually by multiple chromatography of its hydrolysate on silica gel. The IH NMR spectrum of the compund tested by the number of anomeric signals in the 5 ppm region and that of methyl duplicatl" in the 1.20-1.50 ppm region demonstrated the presence of four anomeric forms in the monosaccharide aqueous solution. Differential spectrometry revealed signals of three forms available in the largest amounts. Table 2 presents the values of chemical shifts and coupling constants of the predominant form. The duplicate of triple intensity at 6 = 1.35 ppm confirmed the presence of a 6-deoxy group in the compound tested, while values of 1.95 ppm and 2.13 ppm for 2 protons at C-3 03a,3e = 12.5 Hz), implied the presence of an
Table 1. Rf values of the unknown sugar and other chromatographically highly mobile hexoses occurring in the cell walls of aerobic actinomycetes Solvent system
Type of thinlayer plates
Rf values rhamnose
2-0-methyl- 3-0-methyl- unknown mannose rhamnose hexose
n-Butanol-pyridinewater (6:4:3)
Cellulose Merck, Art. 5716
0.57
0.58
0.74
0.60
n-Butanol-pyridinewater-toluene (10:6:6:1)
Merck, Art. 5716
0.58
0.60
0.76
0.64
Merck, Art. 5716 Silica gel 60 F 254 Merck, Art. 5719
0040
0.57
0.58
0047
0.10
0.17
0.23
0.14
n-Butanol-acetic acid-water (4:1:1) iso-Propanolchloroform-ammonia water (10:10:1)
Tyvelose in Agromyces Cell Walls Table 2. The data of 1H NMR spectroscopy of the monosaccharide tested for the a-pyranose form (d HOD = 4.95 ppm, t = 300 K) 0
Signal
Chemical shift, ppm
Coupling constants, Hz
H-1 (lH) H-2 (lH) H-3a (lH) H-3e (lH) H-4 (lH) H-5 (lH) H-6 (3H)
5.05 4.01 1.95 2.13 3.70 3.95 1.35
(bd, J1,2 = 2.3; J1,3e = 1.0) (m, 12.3a = 3.0; 12.3e = 4.0) (m, ha,4 = 10.5; J3a,3e = 12.5) (m, J3e,4 = 4.0) (m, J4,5 = 9.0) (m, J5,6 = 6.0) (d)
additional3-deoxy group. A large coupling constant of the axial proton at C-3 with a proton in C-4 (10.5 Hz) and a small constant with a proton in C-2 (3 Hz) indicated the manno-configuration of hydroxy groups in the ring. The presence of a distant constant 1,3e = 1.0 Hz) proved the equatorial proton location at C-1 against H-3e (2.13 ppm). Consequently, in the case of 3-deoxy-D-rhamnose the spectrum presented in Table 2 corresponds to the uanomer of the pyranose form (Bock and Pedersen, 1983). The data obtained were also supported by the l3C NMR data. The signals of three anomers corresponding to two pyranose and one furanose forms had the following chemical shifts (OCH3 0H = 49.6 ppm): C-I, 95.2, 93.4,102.8; C2, C-4 and C-5, 67.5, 67.8, 68.6, 68.7, 68.8, 70.5, 76.0, 76.6,82.5; C-3, 32.1, 33.7, 37.5; C-6, 17.5, 17.8, 18.8. The above data suggest that the monosaccharide tested was 3-deoxy-rhamnose (Atlas of mass spectra of organic compounds, 1984). The choice of 3-deoxy-rhamnose epimers (tyvelose and askarylose) was based on the results of optical rotation. The specific rotation of the sugar under study was +22 which was in good agreement with that for tyvelose (Eveleigh, 1983). Thus, the structure of the unknown monosaccharide was established to be 3,6-dideoxy-Darabinohexose (or 3,6-dideoxy-D-mannose, or tyvelose).
a
189
Tyvelose content, its localization in cells and distribution
Comparison of the content of the sugar in hydrolysates of A. cerinus ssp. cerinus VKM Ac-1340 whole cells and native cell walls (Table 3) revealed that galactose, xylose, rhamnose, and tyvelose are cell components of the cell wall and, apparently, originate from a polysaccharide linked to peptidoglycan. The molar ratio of the cell wall sugars in strain VKM Ac-1340 was 1: 1.1: 3.2: 3.8 (galactose-xylose-rhamnose-tyvelose). Other sugars - glucose, mannose, ribose and (in some cases) unidentified sugars (heptose) - are presumably of cytoplasmic origin and/or may originate from a capsular polysaccharide. Tyvelose was found in the cell wall of all five A. cerinus ssp. cerinus strains and in some samples of whole cells of A. ramosus. Its content in whole cell samples of various A. cerinus ssp. cerinus strains grown in a liquid medium for phytopathogenic bacteria under the same conditions can vary considerably. In all A. cerinus ssp. cerinus strains grown under different conditions its content may insignificantly vary. In the A. cerinus ssp. nitratus, A. fucosus ssp. fucosus, and A. fucosus ssp. hyppurates strains tyvelose was not found. In 46 strains of Clavibacter ssp. tested that are with regard to Agromyces strains by their peptidoglycan are similar to Agromyces, no tyvelose could be found. In the cell walls of other Gram-positive bacteria which we have investigated in recent years, no tyvelose has been found, either. According to the literature, tyvelose has been found only in lipopolysaccharides of Gram-negative bacteria Yersinia (Pasteria) pseudotuberculosis (Eveleigh, 1983) as well as Salmonella typhi and S. strasbourg (serogroup D) (Davies, 1960); Kaufmann et al., 1960; Eveleigh, 1983). It is present in the O-antigen and involved in the serological specifity of the bacteria.
Table 3. Quantitative monosaccharide composition of whole cells and cell wall hydrolysates of Agromyces spp. Strains A.ramosus ATCC 25173 T A. cerinus ssp. cerinus VKM Ac-1340T
VKM Ac-1341 VKM Ac-1342 VKM Ac-1343
mannose
xylose
ribose
fucose
rhamnose
tyvelose
heptose
1.5
2.9
7.3
7.5
- b
11.9
tr
1.5
8.3
8.2
6.5
18.1 16.6 7.1
6.5 4.3 2.8
1.1 7.3 0.9* 1.3 0.5 0.4
nd
10.8 29.2 3.2* 8.8 28.5 12.9
6.8 29.2 3.8* 5.1 0.1 0.1
nd
Prep aration
galactose
glucose
wh.c.
o.r
wh.c. C.w. C.w. wh.c. wh.c. wh.c.
4.5 9.9 1.0* 6.7 11.9 5.3
5.1 9.1 6.9
1.5
Designations: a, J-tg/mg of preparation dry weight; b, sugar is absent; tr, trace, presents only in some samples; nd, no data; wh.c., whole cells; c.w., cell walls; *, molar ratio in comparison to galactose.
190
1.1. Maltsev, A.1. Kalinovsky, H.1. Zgurskaya, and L.1. Evtushenko
References Atlas of mass spectra of organic compounds, pp. 66-67. In: Mass spectra of derivative monosaccharides (V. A. Koptiug, ed.). ~ov~sibirsk, Novosibirsk Institute of Organic Chemistry, the Sibenan Branch of the USSR Academy of Sciences 1984 Asano, K., Hiroshi, S., Sano, H., Kawamoto, I.: 3-0-Methylrhamnose: identification and distribution in Catellatospora species and related actinomycetes. Int. J. System. Bact. 39, 56-60 (1989) Bock, K., Pedersen, c.: Carbon-13-magnetic resonance spectroscopy of monosaccharides. Adv. Carbohydr. Chern. Biochem. 41,27-66 (1983) Casida, L. E.: Genus Agromyces, pp. 1329-1331. In: Bergey's Manual of Systematic Bacteriology, Vol. 2 (P. H. A. Sneath, N. S. Mair, M. E. Sharpe, J. C. Holt, eds.). Baltimore, Williams and Wilkins 1986 Davies, D. A. L.: Polysaccharides of Gram-negative bacteria. Adv. Carbohydr. Chern. 15,271-278 (1960)
Davis, M. I., Bil/aspie, A. G., Vidaver, A. K., Harris, R. W.: Clavibacter: a now genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis
subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. Int. J. System. Bact. 34, 107-117 (1984) Easterwood, V. M., Huff, B. J. L.: Carbohydrate analysis by gas chromatography of acetylated aldonitriles. Svensk. Paperstidn. 72, 768-772 (1969) Eveleigh, D.: Microbial monosaccharides and polysaccharides, pp. 3-60. In: Handbook of Microbiology, Vol. 4 (A. I. Laskin, H. A. Lechevalier, eds.). Boca RatonJFL, CRC Press 1983
Evtushenko, L. I., Dobrovolskaya, T. G., Lysak, L. V., Chernyakovskaya, N. F., Zgurskaya, H. I., Adanin, V. M., Ilchenko, V. Ya., Kalakoutskii, L. V.: Actinomycetes with diaminobutyric acid in the cell wall, isolated from soils. Mikrobiologiya 60, 920-925 (1991) (in Russian) Hasegawa, T., Takisawa, M., Tanida, S.: A rapid analysis for chemical grouping of aerobic actinomycetes. J. Gen. Appl. Microbiol. 29, 319-322 (1983) Kaufmann, F., Liideritz, 0., Stierlin, H., Westphal, 0.: Zur Immunochemie der O-Antigene von Enterobacteriaceae. Zbl. Bakt., I. Abt. Orig. 178, 422-449 (1960) Lechevalier, M. P., Lechevalier, H. A.: The chemotaxonomy of actinomycetes, pp. 227-291. In: Actinomycete taxonomy (A. Dietz, D. W. Thayer, eds.). Arlington, Soc. Industrial Microbiology 1980 Lechevalier, M. P., Lechevalier, M. A.: Genus Frankia Brumhorst 1986, 174 AL, pp. 2410-2417. In: Bergey's Manual of Systematic Bacteriology, Vol. 4 (S. T. Williams, M. E. Sharpe, J. G. Holt, eds.). Baltimore, Williams and Wilkins 1989 Pridham, T. G., Gottlieb, D.: The utilisation of carbon compounds by some Actinomycetales as an aid for species determination. J. Bact. 56, 107-114 (1948) Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N., Voyevoda, H. V., Lysak, L. V., Dobrovolskaya, T. G., Kalakoutskii; L. V.: Emended descriotions of the genus Agromyces Casida and Gladhill 1969 and description of the new species and subspecies Agromyces cerinus subsp. cerinus, Agromyces cerinus subsp. nitratus, Agromyces fucosus subsp. fucosus and Agromyces fucosus subsp. hyppuratus. Int. J. System. Bact. (1992, in press)
Dr. ~yudmila I. Evtus~enko, Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences, Pushchino, Moscow RegIOn 142292, RUSSia
Tyvelose in Agromyces Cell Walls 1. 1. MALTSEV1, A. I. KALINOVSKyl, HELEN I. ZGURSKAYA2 , and LYUDMILA I. EVTUSHENK0 2 * 1 2
Pacific Institute of Bioorganic Chemistry, Academy of Sciences, Vladivostok 690 022, Russia Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences, Pushchino, Moscow Region 142 292, Russia
Revision received December 6, 1991
Summary Tyvelose (3,6-dideoxy-D-mannose or 3,6-dideoxy-D-arabinohexose) was found in hydrolysates of whole cells and native cell walls of Agromyces cerinus ssp. cerinus and some whole cell samples of Agromyces ramosus type strain (trace). The sugar was isolated, purified and identified by thin-layer and gas liquid chromatography, mass spectrometry, proton eH) and BC nuclear magnetic resonance (NMR) spectroscopy as well as by optical spectrophotometry . Tyvelose was not found in the cell wall of other actinomycetes and Gram-positive bacteria tested so far.
Key words: Agromyces - Clavibacter - 3,6-Dideoxy-D-arabinohexose - 3,6-Dideoxy-D-mannose - Tyvelose - Cell wall Introduction We recently detected in whole cell hydrolysates of the aerobic actinomycete Agromyces cerinus ssp. cerinus (Evtushenko et ai., 1991; Zgurskaya et ai., 1991) a hexose constituent that was not found previously in actinomycetes and other Gram-positive bacteria. This paper presents its identification as 3,6-dideoxy-D-arabinohexose (or 3,6-dideoxy-D-mannose, or tyvelose). Materials and Methods Bacterial strains and culture conditions. We used fifteen strains
of Agromyces, A. ramosus ATCC 17325 (Casida, 1986) and A.
cerinus ssp. cerinus VKM Ac-1340T, VKM Ac-1341, VKM Ac-1342, VKM Ac-1343, VKM Ac-1344, VKM Ac-1350, A. cerinus ssp. nitratus VKM Ac-1351 T, A. (ucosus ssp. (ucosus VKM Ac-1345 T, VKM Ac- 1346, VKM Ac-1347, VKM Ac-1348, VKM Ac-1349, A. (ucosus ssp. hyppuratus VKM Ac-1352T, VKM Ac-1353 (Zgurskaya et aI., 1991) together with 46 representatives of the genus Clavibacter (Davis et aI., 1984), including the type strains C. michiganensis ssp. michiganensis ICMP 2550 T (= NCPPB 2979 T), C. michiganensis ssp. insidiosus ICMP 2621 T (= NCPPB 1109 T), C. michiganensis ssp. nebraskensis ICMP 3298 T (= NCPPB 2581 T ), C. michiganensis ssp. sepedonicus ICMP 2535 T (= NCPPB 2137T) C. michiganensis ssp. tesselarius ICMP 7221T (= ATCC 33566T), C. iranicus
* Corresponding author
ICMP 3496T (= NCPPB 2253 T), C. rathayi ICMP 2574 T (= NCPPB 2980T), C. tritici ICMP 2626T (= ATCC 11403 T ), obtained from Dr. J. M. Young (Auckland, New Zealand). VKM, All-Union Collection of Microorganisms, Institute of biochemistry and physiology of microorganisms, Puschino, Moskow region, USSR; ICMP, International collection of microorganism from plants, Plant diseases division, DSIR, Auckland, New Zealand; NCPPB, National collection of Plant Pathogenic bacteria, Plantpathology laboratory, Hapenden, Hertsfordshire, U.K. The cultures were grown aerobically in a liquid medium commonly used for phytopathogenic corynebacteria (casein peptone, 10 g; yeast extract,S g; glucose,S g; NaCl, 5 g; distilled water, 1 I) at 30°C for 24 h as well as on solid mineral medium (Pridham and Gottlieb, 1948) with glucose, rhamnose, mannose, galactose, or xylose as carbon source and addition of yeast extract and casamino acids (by 0.1%) containing agar (1,5%). Preparation o( cells and cell walls. The cultures were washed with saline solution (0.8% NaCl) and a mixture of chloroform and methanol (1 : 1, v/v) and air dried. To obtain cell walls, living cells were disrupted using a ball mill with glass beads; the cell \\";llls were separated by differential centrifugation and repeatedly wah sed with water and a chloroform-methanol (1: 1, v/v) mixture and air dried. Chemical analysis o( sugars. The sugar composition of various acid hydrolysates (hydrolysis conditions: 0.5 M HCl, 120 °C, 30 min; 1 M trifluoroacetic acid, 100°C, 1 h; 2 M H 2S0 4 , 100°C, 1 h) was assayed according to the earlier described chromatography methods (Hasegawa et aI., 1983; Lechevalier and Lechevalier, 1980) using Cellulose (Merck, Art. 5716) and
188
1.1. Maltsev, A.1. Kalinovsky, H.1. Zgurskaya, and L.I. Evtushenko
Kieselgel 60 (Merck, Art. 5719) plates as well as by gas liquid chromatography. Acetylated aldonitriles were analyzed by gas chromatography (Easterwood and Huff, 1969). A Tsvet chromatograph (USSR) was used: glass column (2 m X 3 mm LD.)j packed phase, 3% QF on 100/120 mesh Cromatron NAWHMDSj carrier gas argon (60 cm 3/min); the temperature program was 150 0-100/min-220 °C. To identify the unknown sugar, an LKB-9000 gas chromatograph-mass spectrometer was employed with the following characteristics: column (3 m X 3 mm) with 0.1 % QF-1 on a Chromatron NAW-HMDSj carrier gas helium (50 cm 3/min), column temperature program 150o-10olmin-220°Cj ionization voltage, 70 eV; temperature of the ionic source, molecular sefarator and evaporator, 225, 265 and 275°C, respectively. The Hand 13C NMR spectra were recorded by a Bruker WH-250 spectrometer in D2 0 . Optical rotation was measured using a Model 141 Perkin Elmer polarimeter by D-line irradiation in the sodium lamp spectrum.
Isolation and purification of the unknown sugar Dry biomass (5 g) of strain A. cerinus ssp. cerinus VKM Ac-1340 was heated for 1.5 h in 20 ml of 1 M trifluoroacetic acid at 100°C and centrifuged. The pellet was discarded, the supernatant was evaporated and dried under vacuum. Nearly 2 g of the darkbrown precipitate was formed and then dissolved in 5 ml of water with subsequent addition of 60 ml ethanol. The precipitate formed was separated again by centrifugation. After evaporation of the supernatant under vacuum the dry pellet (1 g) was applied to a column with L 40/100 silica gel in the solvent system chloroform:methanol:water (75: 25 : 1, vi v). The separation was controlled by thin-layer chromatography on silica gel plates in the same solvent system. The fractions containing the chromatographically pure monosaccharide were combined and evaporated under vacuum. White powder (22 mg) was obtained.
Results and Discussion
Identification of the unknown sugar
Acid hydrolysis of the preparations and the detection of the unknown sugar. The analysis of various acid hydrolysates of whole cell and native cell wall preparations in some solvent systems revealed a sugar with an Rf exceeding that of rhamnose and differing by its properties from other highly mobile sugars previously found in aerobic actinomycetes (Asano et aI., 1989; Hasegawa et aI., 1983; Lechevalier and Lechevalier, 1986) (Table 1). The sugar gave a brown colour with aniline phthalate. The sugar could not be detected when H 2 S0 4 was used for hydrolysis. (In the method of Lechevalier and Lechevalier (1980) which is often employed for whole cell hydrolysates of actinomycetes H 2 S0 4 is used as acid). The analysis of the sugar acetylated aldonitriles by gas liquid chromatography also revealed a peak with a retention time shorter than that of the rhamnose derivative and differing from those derivatites of other sugars of the standard solution (glucose, galactose, arabinose, mannose, ribose and xylose).
The mass spectrum of the compound obtained by gas liquid chromatography and mass spectrometry was identical to that of paracetate aldonitrile 3,6-dideoxy-hexose (m/z: 212, 184 (100), 142, 129, 128) (Atlas of the mass spectra of organic compounds, 1984). To determine the configuration of its asymmetric centres, the monosaccharide was isolated individually by multiple chromatography of its hydrolysate on silica gel. The IH NMR spectrum of the compund tested by the number of anomeric signals in the 5 ppm region and that of methyl duplicatl" in the 1.20-1.50 ppm region demonstrated the presence of four anomeric forms in the monosaccharide aqueous solution. Differential spectrometry revealed signals of three forms available in the largest amounts. Table 2 presents the values of chemical shifts and coupling constants of the predominant form. The duplicate of triple intensity at 6 = 1.35 ppm confirmed the presence of a 6-deoxy group in the compound tested, while values of 1.95 ppm and 2.13 ppm for 2 protons at C-3 03a,3e = 12.5 Hz), implied the presence of an
Table 1. Rf values of the unknown sugar and other chromatographically highly mobile hexoses occurring in the cell walls of aerobic actinomycetes Solvent system
Type of thinlayer plates
Rf values rhamnose
2-0-methyl- 3-0-methyl- unknown mannose rhamnose hexose
n-Butanol-pyridinewater (6:4:3)
Cellulose Merck, Art. 5716
0.57
0.58
0.74
0.60
n-Butanol-pyridinewater-toluene (10:6:6:1)
Merck, Art. 5716
0.58
0.60
0.76
0.64
Merck, Art. 5716 Silica gel 60 F 254 Merck, Art. 5719
0040
0.57
0.58
0047
0.10
0.17
0.23
0.14
n-Butanol-acetic acid-water (4:1:1) iso-Propanolchloroform-ammonia water (10:10:1)
Tyvelose in Agromyces Cell Walls Table 2. The data of 1H NMR spectroscopy of the monosaccharide tested for the a-pyranose form (d HOD = 4.95 ppm, t = 300 K) 0
Signal
Chemical shift, ppm
Coupling constants, Hz
H-1 (lH) H-2 (lH) H-3a (lH) H-3e (lH) H-4 (lH) H-5 (lH) H-6 (3H)
5.05 4.01 1.95 2.13 3.70 3.95 1.35
(bd, J1,2 = 2.3; J1,3e = 1.0) (m, 12.3a = 3.0; 12.3e = 4.0) (m, ha,4 = 10.5; J3a,3e = 12.5) (m, J3e,4 = 4.0) (m, J4,5 = 9.0) (m, J5,6 = 6.0) (d)
additional3-deoxy group. A large coupling constant of the axial proton at C-3 with a proton in C-4 (10.5 Hz) and a small constant with a proton in C-2 (3 Hz) indicated the manno-configuration of hydroxy groups in the ring. The presence of a distant constant 1,3e = 1.0 Hz) proved the equatorial proton location at C-1 against H-3e (2.13 ppm). Consequently, in the case of 3-deoxy-D-rhamnose the spectrum presented in Table 2 corresponds to the uanomer of the pyranose form (Bock and Pedersen, 1983). The data obtained were also supported by the l3C NMR data. The signals of three anomers corresponding to two pyranose and one furanose forms had the following chemical shifts (OCH3 0H = 49.6 ppm): C-I, 95.2, 93.4,102.8; C2, C-4 and C-5, 67.5, 67.8, 68.6, 68.7, 68.8, 70.5, 76.0, 76.6,82.5; C-3, 32.1, 33.7, 37.5; C-6, 17.5, 17.8, 18.8. The above data suggest that the monosaccharide tested was 3-deoxy-rhamnose (Atlas of mass spectra of organic compounds, 1984). The choice of 3-deoxy-rhamnose epimers (tyvelose and askarylose) was based on the results of optical rotation. The specific rotation of the sugar under study was +22 which was in good agreement with that for tyvelose (Eveleigh, 1983). Thus, the structure of the unknown monosaccharide was established to be 3,6-dideoxy-Darabinohexose (or 3,6-dideoxy-D-mannose, or tyvelose).
a
189
Tyvelose content, its localization in cells and distribution
Comparison of the content of the sugar in hydrolysates of A. cerinus ssp. cerinus VKM Ac-1340 whole cells and native cell walls (Table 3) revealed that galactose, xylose, rhamnose, and tyvelose are cell components of the cell wall and, apparently, originate from a polysaccharide linked to peptidoglycan. The molar ratio of the cell wall sugars in strain VKM Ac-1340 was 1: 1.1: 3.2: 3.8 (galactose-xylose-rhamnose-tyvelose). Other sugars - glucose, mannose, ribose and (in some cases) unidentified sugars (heptose) - are presumably of cytoplasmic origin and/or may originate from a capsular polysaccharide. Tyvelose was found in the cell wall of all five A. cerinus ssp. cerinus strains and in some samples of whole cells of A. ramosus. Its content in whole cell samples of various A. cerinus ssp. cerinus strains grown in a liquid medium for phytopathogenic bacteria under the same conditions can vary considerably. In all A. cerinus ssp. cerinus strains grown under different conditions its content may insignificantly vary. In the A. cerinus ssp. nitratus, A. fucosus ssp. fucosus, and A. fucosus ssp. hyppurates strains tyvelose was not found. In 46 strains of Clavibacter ssp. tested that are with regard to Agromyces strains by their peptidoglycan are similar to Agromyces, no tyvelose could be found. In the cell walls of other Gram-positive bacteria which we have investigated in recent years, no tyvelose has been found, either. According to the literature, tyvelose has been found only in lipopolysaccharides of Gram-negative bacteria Yersinia (Pasteria) pseudotuberculosis (Eveleigh, 1983) as well as Salmonella typhi and S. strasbourg (serogroup D) (Davies, 1960); Kaufmann et al., 1960; Eveleigh, 1983). It is present in the O-antigen and involved in the serological specifity of the bacteria.
Table 3. Quantitative monosaccharide composition of whole cells and cell wall hydrolysates of Agromyces spp. Strains A.ramosus ATCC 25173 T A. cerinus ssp. cerinus VKM Ac-1340T
VKM Ac-1341 VKM Ac-1342 VKM Ac-1343
mannose
xylose
ribose
fucose
rhamnose
tyvelose
heptose
1.5
2.9
7.3
7.5
- b
11.9
tr
1.5
8.3
8.2
6.5
18.1 16.6 7.1
6.5 4.3 2.8
1.1 7.3 0.9* 1.3 0.5 0.4
nd
10.8 29.2 3.2* 8.8 28.5 12.9
6.8 29.2 3.8* 5.1 0.1 0.1
nd
Prep aration
galactose
glucose
wh.c.
o.r
wh.c. C.w. C.w. wh.c. wh.c. wh.c.
4.5 9.9 1.0* 6.7 11.9 5.3
5.1 9.1 6.9
1.5
Designations: a, J-tg/mg of preparation dry weight; b, sugar is absent; tr, trace, presents only in some samples; nd, no data; wh.c., whole cells; c.w., cell walls; *, molar ratio in comparison to galactose.
190
1.1. Maltsev, A.1. Kalinovsky, H.1. Zgurskaya, and L.1. Evtushenko
References Atlas of mass spectra of organic compounds, pp. 66-67. In: Mass spectra of derivative monosaccharides (V. A. Koptiug, ed.). ~ov~sibirsk, Novosibirsk Institute of Organic Chemistry, the Sibenan Branch of the USSR Academy of Sciences 1984 Asano, K., Hiroshi, S., Sano, H., Kawamoto, I.: 3-0-Methylrhamnose: identification and distribution in Catellatospora species and related actinomycetes. Int. J. System. Bact. 39, 56-60 (1989) Bock, K., Pedersen, c.: Carbon-13-magnetic resonance spectroscopy of monosaccharides. Adv. Carbohydr. Chern. Biochem. 41,27-66 (1983) Casida, L. E.: Genus Agromyces, pp. 1329-1331. In: Bergey's Manual of Systematic Bacteriology, Vol. 2 (P. H. A. Sneath, N. S. Mair, M. E. Sharpe, J. C. Holt, eds.). Baltimore, Williams and Wilkins 1986 Davies, D. A. L.: Polysaccharides of Gram-negative bacteria. Adv. Carbohydr. Chern. 15,271-278 (1960)
Davis, M. I., Bil/aspie, A. G., Vidaver, A. K., Harris, R. W.: Clavibacter: a now genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis
subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. Int. J. System. Bact. 34, 107-117 (1984) Easterwood, V. M., Huff, B. J. L.: Carbohydrate analysis by gas chromatography of acetylated aldonitriles. Svensk. Paperstidn. 72, 768-772 (1969) Eveleigh, D.: Microbial monosaccharides and polysaccharides, pp. 3-60. In: Handbook of Microbiology, Vol. 4 (A. I. Laskin, H. A. Lechevalier, eds.). Boca RatonJFL, CRC Press 1983
Evtushenko, L. I., Dobrovolskaya, T. G., Lysak, L. V., Chernyakovskaya, N. F., Zgurskaya, H. I., Adanin, V. M., Ilchenko, V. Ya., Kalakoutskii, L. V.: Actinomycetes with diaminobutyric acid in the cell wall, isolated from soils. Mikrobiologiya 60, 920-925 (1991) (in Russian) Hasegawa, T., Takisawa, M., Tanida, S.: A rapid analysis for chemical grouping of aerobic actinomycetes. J. Gen. Appl. Microbiol. 29, 319-322 (1983) Kaufmann, F., Liideritz, 0., Stierlin, H., Westphal, 0.: Zur Immunochemie der O-Antigene von Enterobacteriaceae. Zbl. Bakt., I. Abt. Orig. 178, 422-449 (1960) Lechevalier, M. P., Lechevalier, H. A.: The chemotaxonomy of actinomycetes, pp. 227-291. In: Actinomycete taxonomy (A. Dietz, D. W. Thayer, eds.). Arlington, Soc. Industrial Microbiology 1980 Lechevalier, M. P., Lechevalier, M. A.: Genus Frankia Brumhorst 1986, 174 AL, pp. 2410-2417. In: Bergey's Manual of Systematic Bacteriology, Vol. 4 (S. T. Williams, M. E. Sharpe, J. G. Holt, eds.). Baltimore, Williams and Wilkins 1989 Pridham, T. G., Gottlieb, D.: The utilisation of carbon compounds by some Actinomycetales as an aid for species determination. J. Bact. 56, 107-114 (1948) Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N., Voyevoda, H. V., Lysak, L. V., Dobrovolskaya, T. G., Kalakoutskii; L. V.: Emended descriotions of the genus Agromyces Casida and Gladhill 1969 and description of the new species and subspecies Agromyces cerinus subsp. cerinus, Agromyces cerinus subsp. nitratus, Agromyces fucosus subsp. fucosus and Agromyces fucosus subsp. hyppuratus. Int. J. System. Bact. (1992, in press)
Dr. ~yudmila I. Evtus~enko, Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences, Pushchino, Moscow RegIOn 142292, RUSSia