Electron Microscopy of Alkaline Phosphatase of Escherichia Coli

Abstract

A method is described for determining the localization of alkaline phosphatase in the cells of E. coli B with the electron microscope. Enzyme activity, determined by deposition of inorganic phosphate, is located in the exterior layer of the cell wall.Electron microscopy is of great importance in the study of the relationship of enzymes and cell ultrastructures and this application has permitted the localization of some esterases and dehydrogenases (1,4,11). A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.The present paper concerns the distribution of alkaline phosphatase in the cells of Escherichia coli B. Materials a n d MethodsThe cells used were E . coli B. An 18-hour culture of E. coli B on nutrient agar was inoculated into a salts medium having limited inorganic phosphate (17) and grown for 24 to 48 hours. All the salts were recrystallized. Sodium P-glycerophosphate for determination of phosphatase was obtained from the Merck Company. No trace of free orthophosphate was found in this preparation.Coloration.-After growth, the cells were centrifuged and placed in a glycerophosphate medium (3) a t pH 9.0. The incubation was continued a t 37 OC for 10 minutes to 2 hours. After that the cells were again centrifuged and suspended in a 2y0 cobalt nitrate soiution, b u t in some experiments, suspension in cobalt solution was omitted. After 2-3 minutes the cell suspension in cobalt solution was centrifuged a t 3000 r.p.m. for 3 minutes and the supernatant was placed on formvar-coated grids for electron microscopy.Ultrathin sections.-The material obtained after cobalt nitrate treatment was fixed according to Ryter et al. (13), dehydrated in alcohols, and embedded in the mixture of butyl-and methyl-metacrylates (4: 1). The polymerization was performed a t 55' for 24-48 hours. The sections were obtained on the "Ultratome 4800" with glass knives and collected on formvar-covered copper grids strengthened with carbon. The material obtained was contrasted in 5yo uranyl acetate solution a t room temperature for 1 hour.Electron microscopy.-The preparations of whole cells and sections were looked over in UEMV-100B electron microscope a t 50 and 75 kV with a 40-p objective and recorded with photoplates MR. Electron optical magnification, 8000-62,000 X.

Acid and alkaline phosphatases of Capnocytophaga species. I. Production and cytological localization of the enzymes

“…In Capnocytophaga, however, in addition to a periplasmic location, AcP and A1P were localized at the cell surface. We believe that this surface location is not an artifact because (i) localization by modified Gomori and immunoferritin in capnobacteria was similar in mid-and late-logarithmically grown cells with only cells in late-stationary phase showing an obvious change in location with slightly more product occurring at the cell surface; (ii) aldehyde pretreatment of E. coli (Kushnarev and Smirnova 1966;Wetzel et al 1970) and P. aeruginosa (Cheng et al 1970b) resulted in a periplasmic space to cell surface shift in Alp reaction product. However, formaldehyde prefixation of Capnocytophaga did not induce a similar shift of product indicating the alteration of membrane permeability by formaldehyde does not alter phosphatase location.…”

Localization of phytase inSelenomonas ruminantiumandMitsuokella multiacidusby transmission electron microscopy

“…H3-22 (McLean et al 1985). Alkaline phosphatase has been localized by electron microscopy within the outer membrane of E. coli (Kushnarev and Smirnova 1966), and in the periplasmic space in Pseudomonas aeruginosa, Bacteroides succinogenes, and Bacteroides ruminicola (now Fibrobacter succinogenes (Montgomery et al 1988) and Prevotella ruminicola (Shah and Collins 1990), respectively), and in M. elsdenii (Cheng et al 1970;Cheng and Costerton 1973). This paper investigates the cell-associated localization of the phytase activity of two ruminal bacteria, S. ruminantium JY35, and M. multiacidus 46/5(2), as well as the E. coli pSrP.2 strain expressing a heterologous phytase gene from S. ruminantium JY35; and describes a simple technique for electron microscopic localization of bacterial phytase.…”

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

“…The barrier to the action and free release of the enzymes could, however, be explained as well if the enzymes were attached to or embedded in the cell wall membrane.Efforts have been made to localize the surface enzymes by combining cytochemical and electron microscope techniques. Some studies indicate that wall-associated phosphatases are present in the periplasmic space (8,26), others indicate that they are present at the cell surface (15,20). The pattern of localization obtained appears to be dependent upon the technique used (16,23,26).…”