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Actinomycetes
University of Udine, Mycology Department
ISSN: 0732-0574
Vol. 6, Num. 3, 1995

Actinomycetes 1995 Vol. 6, Part 3

Fatty acid composition of nocardiae

M. Tseng

Food Industry Research & Development Institute, Hsinchu, Taiwan, Republic of China

Code Number: AC95014 Sizes of Files: Text: 15K No associated graphics

Abstract. Fatty acid composition of thirty Nocardia species and subspecies has been analysed by gas liquid chromatography and organisms were accordingly divided into two groups. No mycolic or tuberclostearic acid, were found in a subgroup of organisms and their taxonomic position is discussed.

According to genetic and phenotypic data, the genus Nocardia appears heterogeneous (Lechevalier and Lechevalier, 1970; Mordarski et al., 1972; Kurup and Schmitt, 1973). In the present study, the fatty acid compositions of thirty Nocardia species and subspecies deposited in the Culture Collection and Research Center (CCRC) of Taiwan, Republic of China is reported.

Materials and Methods

Strains. Organisms used in the study are listed in Table 1.

Fatty acid analysis. Strains were incubated on tryptic soy broth (BBL) and incubated at 28 C for 2 to 8 dd. N.thermoflava, strain CCRC 13738, was grown at 45 C. Forty mg of biomass were treated with 1ml of NaOH-methanol at 100 C, methylated with 2ml of 6 N HCl-methanol at 80 C, extracted with 1.25ml of hexane-methyl-tert butyl ether and washed with 3ml of NaOH. The fatty acid methyl esters were analysed by gas liquid chromatography (HP 5890 series II, Hewlett-Packard Co., USA), equipped with a fused-silica capillary column, a flame ionization detector and an HP 3396 II integrator. Gas flow rate was ca. 400ml/min for air, 30ml/min for hydrogen, and 30ml/min for nitrogen. Temperatures were set to 250 C at the injection port and to 300 C at the detector. Oven temperature was increased from 170 C to 270 C at a rate of 5 C/min. Data were analysed according to Miller (1984), using a MIS software package (Microbial ID, Inc., Newark, USA).

Analysis of mycolic acid. Strains were incubated on yeast-glucose broth (yeast extract 10.0g/l, glucose 10.0g/l, pH 7.0) for 4 to 7 dd. Cells were harvested by centrifugation (6,000 rpm, 10 min), and lyophilised. Cells (0.5g) were hydrolysed with 10% KOH-methanol at 100 C. The hydrolysate was acidified with 6 N HCl (pH < 2.0). Free fatty acids and mycolic acid were extracted with n-hexane and methylated with benzene methanol-H2SO4 (10:20:1, v/v) at 100 C. Following addition of 1ml of H2O, fatty acid and methyl esters were extracted with 4ml of n-hexane and separated by thin layer chromatography, using n-hexane-diethyl ether (4:1, v/v) on silica gel (Kieselgel 60, Merck). Spots were visualised with iodine vapours (Komagata and Suzuki, 1987).

Results and Discussion

Fatty acid composition was determined on cells harvested at different times. Data obtained after 4dd incubation were more stable than after 2 or 8dd (data not shown).

Organisms tested can be separated into two groups. Group 1 includes 21 strains (Table 2), characterised by straight-chain fatty acids, tuberclostearic acid (TBSA) but no branched-chain fatty acids (Lechevalier, 1977).

Strains of Group 2 have a variable fatty acid composition, showing straight-chain and/or branched-chain fatty acids with or without TBSA. On this basis a further subdivision is possible. In Subgroup A 'N.carnea', CCRC 13693, and 'N.flavorosea' subsp. 'fusca', CCRC 13730, the major components are represented by straight-chain (nC17:1) and branched-chain (isoC16:0, isoC15:0) fatty acids. A small (5%) amount of TBSA is also present.

None of the remaining seven strains (Subgroup B) contain TBSA (Table 3). Straight- (nC17:1) and branched-chain (isoC16:0) fatty acids are the major components of 'N.diaphanozonaria', CCRC 13744, 'N.thermoflava', CCRC 13738, and 'N.uniformis' subsp. 'tsuyamanensis', CCRC 13314.

--------------------------------------------------------------
  CCRC            Organism                 Origin
  number
-------------------------------------------------------------- 

13749  'N. argentinensis'             ATCC 31306
13380   N. asteriodes                 JCM 3384T(=ATCC 19247)
13389   N. brevicatena                JCM 3029T(=ATCC 15333)
13752  'N. canicruria'                ATCC 17896
13693   N. carnea                     DSM 43397T(=ATCC 6847)
13753  'N. corallina' subsp. 'taoka'  ATCC 31338
13744  'N. diaphanozonaria'           JCM 3208T
13722  'N. farcinica'                 NCTC 11134T
13725  'N. flavorosea'                NRRL B-16176T(=JCM 3332)
13730  'N. flavorosea' subsp. 'fusca  IFO 14342
13734  'N. fusca'                     IFO 14340
13731  'N. gardneri'                  IFO 3385
13750  'N. interforma'                ATCC 21072
12359  'N. italica'                   NCIMB 9386(=DSM 43191)
13732  'N. mexicana'                  IFO 3927
13724  'N. nitrificans'               NRRL B-1664(=JCM 4134)
13746   N. nova                       JCM 6044w(=ATCC 33726)
13751  'N. paraffinae'                ATCC 21509
13694   N. petroleophila              DSM 43193T(=ATCC 15777)
13733   N. pinensis                   IFO 15059T
11086  'N. pseudosporangifera'        IAM 0501
12441  'N. salmonicida'               JCM 4826(=ATCC 27463)
13735  'N. salmonicolor' subsp. 
'aurantiaca'                          IFO 14426
13745   N. seriolae                   JCM 3360T(=ATCC 43993)
13738  'N. thermoflava'               IFO 14333
13612   N. transvalensis              JCM 3319T
13314  'N. uniformis' subsp. 
'tsuyamanensis'                       JCM 3279(=ATCC 21806)
13368  'N. uniformis' subsp. 
'uniformis'                           JCM 3224(=IFO 13702)
13695   N. vaccinii                   DSM 43285(=ATCC 11092)
13736  'N. violaceofusca'             IFO 144
-------------------------------------------------------------

Table 1. Origin of the Nocardia cultures. Names in inverted commas are not on the Approved Lists of Bacterial Names (Skerman et al., 1980). ATCC, American Type Culture Collection, Rockville, USA; CCRC, Culture Collection and Research Center, Food Industry Research and Development Institute, Taiwan, Rep. China; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; JCM, Japan Collection of Microorganism; IAM, Institute of Applied Microbiology, University of Tokyo, Japan; IFO, Institute of Fermentation, Osaka, Japan; NCIMB, National Collection of Industrial and Marine Bacteria, UK; NCTC, National Collection of Type Cultures, Public Health Laboratory, UK; NRRL, ARS Culture Collection, Northern Regional Research Center, USA. T: type strain.

-------------------------------------------------------------- FATTY CCRC STRAIN NUMBER ACID (%) -------------------------------------------------------------- 13749 13380 13389 13752 13753 13722 13725 13734 -------------------------------------------------------------- nC14:0 0.75 0.96 4.95 2.74 1.13 0.85 1.11 nC15:0 3.38 3.68 6.80 5.41 6.10 2.48 0.55 nC16:0 28.93 24.32 19.75 23.15 19.37 31.59 28.85 34.18 nC17:0 2.64 5.08 9.66 2.26 5.25 3.70 1.04 1.65 nC18:0 3.11 7.44 6.74 0.84 6.15 12.04 8.14 nC19:0 0.49 2.16 7.29 1.17 1.13 nC20:O 0.64 6.26 1.44 0.49 1.35 nC15:1 0.23 1.00 0.30 nC16:1 1.08 1.08 0.61 14.43 nC17:1 1.93 1.82 8.06 6.88 6.64 0.43 0.70 0.66 nC18:1 12.40 2.79 7.01 4.81 5.48 6.03 26.49 18.18 nC19:1 0.65 3.42 2.21 1.72 2.23 1.56 nC20:l 0.73 3.08 3.16 nC20:2 nC20:4 3.33 0.98 10MeC16:0 1.95 2.11 10MeC17:0 1.20 1.13 11.00 3.35 5.13 0.51 10MeC18:0 27.24 27.64 18.14 13.95 21.32 26.01 16.60 17.89 nC16:0/ 15i0H 17.77 16.97 8.47 12.25 14.43 15.89 5.03 -------------------------------------------------------------- FATTY CCRC STRAIN NUMBER ACID (%) 13731 13732 13746 13751 13733 11086 12441 13735 -------------------------------------------------------------- nC14:0 1.37 0.83 0.67 1.04 1.49 2.37 nC15:0 0.52 0.85 1.27 1.20 5.77 4.89 nC16:0 35.56 23.76 36.01 29.26 22.34 30.77 28.20 24.55 nC17:0 0.38 1.76 3.39 0.63 9.56 4.51 7.58 2.60 nC18:0 3.17 9.67 6.84 6.33 14.49 12.21 nC19:0 0.41 1.35 nC20:0 1.52 0.38 0.37 0.80 nC15:1 0.42 nC16:1 1.11 0.48 4.10 1.10 1.04 nC17:1 0.53 0.62 1.18 7.31 nC18:1 38.16 4.94 18.03 24.37 19.04 15.29 10.76 10.55 nC19:1 0.53 1.90 0.54 0.66 9.88 9.19 0.87 nC20:l 7.32 0.76 3.67 0.37 nC20:2 0.46 11.85 nC20:4 1.25 2.72 10MeC16:0 0.21 1.31 10MeC17:0 0.79 0.70 4.16 10MeC18:0 4.12 29.20 19.36 19.38 15.05 28.39 17.19 15.91 nC16:0/ 15iOH 13.45 14.39 10.39 10.19 5.28 8.84 9.46 17.88 -------------------------------------------------------------- FATTY CCRC STRAIN NUMBER ACID (%) 13745 13612 13695 13736 13368 -------------------------------------------------------------- nC14:0 1.28 1.83 0.74 0.58 nC15:0 14.42 2.75 4.44 0.77 5.02 nC16:0 23.96 36.85 33.13 33.92 24.86 nC17:0 4.43 3.23 4.09 3.22 nC18:0 2.70 2.26 13.36 1.16 nC19:0 1.53 0.52 nC20:0 3.15 nC15:1 6.8 nC16:1 0.77 1.85 0.30 nC17:1 7.41 2.70 5.88 3.25 nC18:1 15.59 11.38 10.18 6.79 12.62 nC19:1 3.53 0.50 3.08 nC20:1 1.34 1.50 nC20:2 1.04 2.08 nC20:4 1.05 10MeC16:0 0.61 10MeC17:0 5.38 2.96 3.62 0.45 2.81 10MeC18:0 5.36 24.63 28.94 21.96 24.53 nC16:0/ 15i0H 10.50 6.67 5.94 21.96 13.86 ------------------------------------------------------------ Table 2. Fatty acid composition of Group 1 nocardiae. ------------------------------------------------------------ FATTY ACID (%) CCRC STRAIN NUMBER 13693 13730 13744 13738 13314 13750 13724 13694 12359 nC14:0 0.42 0.59 0.22 0.30 0.21 2.09 nC15:0 0.77 1.22 4.02 0.85 5.80 5.59 0.58 2.00 1.39 nC16:0 7.48 2.30 2.96 2.54 2.93 0.86 1.09 9.69 3.99 nC17:0 1.63 9.23 1.89 4.51 1.18 0.40 2.04 0.72 nC18:0 1.56 0.54 0.55 nC15:1 0.66 1.68 0.99 4.17 5.06 1.08 nC16:1 3.33 9.09 1.83 4.86 6.12 1.34 4.07 7.43 0.54 nC17:1 16.47 11.99 15.91 19.02 20.93 3.78 6.65 2.11 0.51 nC18:1 6.37 1.26 0.58 1.51 2.05 IsoC11:0 1.08 IsoC12:0 0.30 5.90 IsoC13:0 0.15 1.12 0.18 IsoC14:0 0.36 2.41 4.23 0.46 8.29 11.3 3.25 3.71 1.20 IsoC15:0 1.49 42.29 8.46 2.21 5.18 20.49 10.89 17.52 8.36 IsoC16:0 40.38 3.68 41.49 44.21 15.54 32.47 39.39 37.35 14.07 IsoC17:0 4.13 4.14 1.42 5.56 3.83 3.74 4.86 6.60 IsoC18:0 0.90 2.82 IsoC16:1 5.03 1.76 0.40 7.19 4.26 14.22 3.60 0.39 IsoC17:1 0.71 5.47 1.23 1.68 7.33 3.66 AnC13:0 1.86 AnC15:0 7.87 1.07 3.12 7.16 0.77 33.56 AnC17:0 2.34 2.67 2.14 3.97 2.49 1.78 3.55 1.35 21.21 AnC17:1 0.23 0.38 0.41 2.90 10MeC16:0 0.25 6.27 10MeC17:0 0.76 0.83 1.12 1.59 0.52 1.75 10MeC18:0 2.74 1.36 CycloC17:0 0.89 nC16:0/15iOH 1.85 16.97 1.55 --------------------------------------------------------------

Table 3. Fatty acid composition of Group 2 nocardiae.

'N.interforma', CCRC 13750, 'N.nitrificans', CCRC 13724, and 'N.petroleophila', CCRC 13694, contain mainly branched-chain fatty acids. IsoC16:0, anteisoC15:0 and anteisoC17:0 are the major fatty acids of 'N.italica', CCRC 12359.

All strains, with the exception of those belonging to Subgroup B, contain mycolic acid.

On the basis of the absence of mycolic acid (Lechevalier, 1989), it is suggested that the seven organisms of Subgroup B be excluded from the genus Nocardia. Further work however is needed to clarify their taxonomic identity.

ACKNOWLEDGEMENTS. This research was supported by a grant from the Ministry of Economic Affairs, Taiwan. The author wishes to thank Drs.W.-S.Chu and J.-S.Way for reviewing the manuscript and the Japan Collection of Microorganisms and the Institute of Fermentation, Osaka, for supplying the strains.

REFERENCES

Komagata, K. & K.Suzuki (1987). Lipid and cell-wall analysis in bacterial systematics. Meth. Microbiol., 19: 161-207

Kurup, P.V. & J.A.Schmitt (1973). Numerical taxonomy of Nocardia. Can.J.Microbiol., 19: 1035- 1048

Lechevalier, H.A. (1989). Nocardioform actinomycetes. In: S.T.Williams, M.E.Sharpe & J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, London, Vol. 4, pp. 2348-2404

Lechevalier, M.P. (1977). Lipids in bacterial taxonomy-a taxonomist's view. CRC Crit.Rev. Microbiol., 5: 109-210

Lechevalier, M.P. & H.A.Lechevalier (1970). Composition of whole cell hydrolysates as a criterion in the classification of aerobic actinomycetes. In: H.Prauser (ed.) The Actinomycetales. Veb G.Fisher, Jena, pp. 311-316

Miller, L. (1984). Gas-liquid chromatography of cellular fatty acid as a bacterial identification aid. HP Application Note: 228-237

Mordarski, H., M.Mordarski & M.Goodfellow (1972). Chemotaxonomic characters and classification of some nocardioform bacteria. J.gen.Microbiol., 71: 77- 86

Skerman, V.B.D., V.McGowan & P.H.A. Sneath (1980). Approved lists of bacterial names. Int.J.Syst.Bacteriol., 30: 225-420

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