Escherichia coliK99 pili are composed of one subunit species

Abstract

1. INTRODUCTION 2.2. Electrophoretic procedures K99 is a pilus found on most, if not all, ente- rotoxigenic Escherichia co# (ETEC) that cause di- arrhea in neonatal calves [1-6] and on up to one third of all ETEC that cause diarrhea in pigs [7,8]. In vivo, K99 promotes colonization of the small intestine by facilitating adhesion of ETEC to the mucosa of the small intestine [9,10]. K99 facilitates adhesion by bridging the space between bacterium and the putative mucosal receptor. K99 has been purified and shown to be a protein polymer composed of subunits of two dif- ferent Mrs: 22500 and 29500 [11]. A small amount of lipid may also be associated with K99. The objective of this manuscript is to report that K99 is composed of only a single subunit species even though two different molecular weight species are observed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. 2. MATERIALS AND METHODS 2.1. Purification of K99 K99 was purified from E. coli K12 strain 1474 as previously described [11] except that the pili were extracted from whole cells using 2 M urea in 0.05 M sodium phosphate (pH 7.2) instead of 1 M NaC1 in the same buffer. SDS-5% polyacrylamide continuous gels were prepared and used according to the procedure of Weber and Osborn [12]. SDS-11, 15 and 20% polyacrylamide discontinuous gels were prepared and used according to the procedure of Lugten- berg [13]. Samples were denatured by boiling 5 min in 2% SDS. When required, 1% 2-mercaptoethanol (MetOH) was included in the denaturation solu- tion. Also when required, 1% MetOH was included in the upper electrophoresis buffer. All gels were stained with Coomassie brilliant blue by the proce- dure of Fairbanks [14]. 2.3. Amino-terminal amino acid determination The procedure of Gros and Labousse [15] was used to dansylate purified K99 with the following modifications. Dansyl chloride was dissolved in acetone and the reaction mixture was adjusted to a final concentration of 8 M urea and 1% SDS. The dansylation reaction was allowed to proceed for 16 h at room temperature after which the protein was precipitated and washed twice with 10% tri- chloroacetic acid. The dansylated K99 was hydro- lyzed for 4 h at 100°C in vacuo with 6 N HC1. The hydrolysate was dried over NaOH, extracted with water (pH 3.5):ether (1 : 1) and both phases were dried. The residue from each phase was disgolved in pyridine and chromatographed in two dimen- sions on silica gel-thin layer plates [15].