Tlie rate of incorporation of [3H]diaminopimelate into tlie lipoprotein attachment sites was compared to tlie rate of its incorporation into total acid-insoluble and dodecylsulpliate-insoluble murein. In contrast to earlier findings, we could detect no significant difference in these rates of incorporation, suggesting that tlie lipoprotein molecules are not specifically excluded from tlie murein growth sites.The amount of lipoprotein bound to the murein increased when the cells entered the stationary phase of growth.The cell wall of gram-negative bacteria consists of a murein layer and a outer membrane. The outer membrane contains a lipoprotein which occurs either in the free form or bound covalently to the murein layer [I -31. The conversion of the free lipoprotein to tlie murein-bound form provides a link between the processes of synthesis and assembly of outer membrane proteins on tlie one hand and the synthesis and Ltssembly of the murein layer on the other hand.We have recently described the kinetics of the conversion of the free to the bound lipoprotein using cells labeled with [35S]methio~iine [4]. Thereby we have developed some experimental techniques for the analysis of the bound form of the lipoprotein that could also be used in case of cells labeled in tlie murein with [3H]diaminopimelic acid. This opens up tlie possibility of studying some aspects of murein synthesis at the lipoprotein attachment sites.There is only one detailed report dealing with these aspects. Braun and Wolff [5] have shown that it takes several generations of growth until the amount of lipoprotein on newly made murein has reached its steady-state value. The interpretation of these results poses coiisiderable problems, since it is difficult to imagine how exponentially growing cells, that double their total cell mass (and thus their content of murein) in a given amount of time, do not in the same time double their amount of lipoprotein on this new murein.Given the above and the availability of suitable techniques to study tlie murein around lipoprotein attachment sites, we found it worthwhile to reexamine the work of Braun and Wolff. Tlie results are presented in this paper and will show that we arrive at conclusions different from those of Braun and Wolff. MATERIALS AND METHODS Organism, Growth Conditions, Ruclioartive Labeling and Sampling of CellsIn this study we used the lysine-requiring, diaminopimelate auxotroph Esclzerichiu coli W7 [6], which was grown in miniinal medium [7] with 0.5 % glucose as carbon source. The medium was supplemented with lysine (50 pg/ml) and ~~-2 , 6 -diaminopimelate (Fluka AG, Buchs SG, Switzerland) as indicated in the text.Labeling with [3H]diaminopimelate (spec. act. 1.1 Ci/ mmol) or [35S]methionine (spec. act. about 1000 Ci/mmol) was performed as described in the text. Samples of 2 mi were withdrawn from the growing cultures, 50 pl (in duplicate) were used for the determination of the amount of acidprecipitable label [4] and the remaining sample was mixed with 300 p1 glycerol and frozen in liquid...

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