NOTES: Two-Dimensional Thin-Layer Chromatography for Amino Acid Analysis of Bacterial Cell Walls

Abstract

Good separation of bacterial cell wall amino acids, including the isomers of diaminopimelic acid, was obtained by two-dimensional thin-layer chromatography, using commercial cellulose-coated aluminum sheets with isopropanol-acetic acid-water (75: 10: 15, vol/vol) and methanol-pyridine-10 N hydrochloric acid-water (64:8:2: 14, vol/vol) as the fist and second solvents, respectively. Cell wall analysis is an established chemotaxonomic method in bacterial systematics. The analyses can be made at various levels of technological sophistication, depending upon the choice, skill, and resources of the operator. Most systematic work still relies upon paper chroma

Proposal of Mycetocola gen. nov. in the family Microbacteriaceae and three new species, Mycetocola saprophilus sp. nov., Mycetocola tolaasinivorans sp. nov. and Mycetocola lacteus sp. nov., isolated from cultivated mushroom, Pleurotus ostreatus.

“…Cell walls were prepared from about 500 mg (dry wt) bacterial cells as described by Schleifer & Kandler (1972). Amino acids in the acid hydrolysate of the cell walls were identified by two-dimensional ascending chromatography on cellulose TLC plates (Tokyo Kasei) by the method of Harper & Davis (1979) and by HPLC as their phenylthiocarbamoyl derivatives with a model Shimadzu LC-6AD HPLC apparatus, according to the manufacturer's instructions. Glycolate tests were performed by the method of Uchida et al (1999).…”

Paenibacillus cisolokensis sp. nov., isolated from litter of a geyser

“…Briefly, dry cells were mechanically disrupted with an ultrasonic oscillator, and precipitates obtained by centrifuging at 18 000 r.p.m for 30 min were heated at 100 C for 40 min in 3 % SDS solution, washed with warm water three times and freeze-dried. The amino acid composition of complete cell-wall hydrolysates was determined by developing preparations on cellulose TLC plates (Tokyo Kasei), using two-dimensional descending chromatography as described by Harper & Davis (1979). Menaquinones were extracted as described by Collins et al (1977) from lyophilized cells grown on ISP 2 at 50 C for 3 days and analysed by HPLC.…”

Four novel Arthrobacter species isolated from filtration substrate

“…For two dimensional TLC, 2-propanol/acetic acid/water (75 : 10 : 15, v/v) was used in the first dimension and methanol-pyridine/10 M hydrochloric acid/water (64 : 8:2 : 14, v/v) was used for the second dimension. The amino acids of the cell-wall peptidoglycan were determined according to the methods of Schleifer & Kandler (1972) and Harper & Davis (1979). The cellular fatty acids were extracted according to the protocol of the MIDI system (Microbial ID Inc.).…”