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Chapter 3

Chemical Composition of Escherichia coli

FREDERICK C. NEIDHARDT and H. EDWIN UMBARGER

 

INTRODUCTION

The need for determining the chemical composition of bacterial cells, especially that of Escherichia coli, has long been recognized. In the modern era, notable measurements of the total composition of this organism were made in 1946 by Taylor (16) and in the early 1950s by a group of biophysicists at the Department of Terrestrial Magnetism of the Carnegie Institution of Washington (13). The latter group, making full use of then newly available radioisotopes, mounted a monumental study of the composition of E. coli, its metabolism, and its control of biosynthetic pathways. Their report (13) was used extensively for nearly four decades. It has been such a valuable sourcebook and guide that it was often called the E. coli bible.

The possibility of an accurate inventory of the molecules of the E. coli cell is greater today than it was in the past. The chemical structures of the more complex components of the cell are closer to being known. Advances have been made in both the isolation of cellular structures and the resolution of complex mixtures of cellular molecules by gas chromatography, high-pressure liquid chromatography, and two-dimensional gel electrophoresis.

The value of an inventory is greater now as well. Most of the fueling and biosynthetic pathways of the E. coli cell have been described, and the general mechanisms for the synthesis of the major macromolecules are reasonably well understood. Therefore, it is possible to calculate flow through pathways and to estimate growth requirements for energy, reducing power, and metabolites under defined conditions (10, 17; chapter 107 of this book). Such calculations help uncover areas in which knowledge is skimpy (energy costs for assembly, processing, transport, and proofreading, for example) and can turn up discrepancies that otherwise would lie hidden. The single most important value of a chemical inventory of the E. coli cell, however, is that it frequently provides the basis for in vivo tests of theories of cell growth and regulation (see references 4 and 10).

 

PROBLEMS IN SPECIFYING CELL SIZE AND COMPOSITION

Aside from technical difficulties inherent in various analytical procedures—and there are many—there are some problems uniquely related to the nature of bacteria. Specifically, these include their size and compositional variation and the heterogeneity of growing cell populations.

It is obviously meaningless to talk about the size or composition of a bacterial cell without specifying the strain, the growth conditions, and the phase of growth. The stationary phase (at least in its early stages of development) is not a unique state, nor are its various developmental stages as easily reproducible in the laboratory as is the exponential phase. Culture phases that are transitional between different stages in growth and between different states of balanced growth, though of great interest to physiologists, are particularly difficult to reproduce for quantitative characterization. Therefore, measurements intended to be compared with those made by others should ideally be made on cultures in steady-state, exponential growth. At the risk of pedantry, it should be pointed out that phrases such as "early-log-phase culture" or "late-log-phase culture" are simply confessions that the investigator has not followed with care the prescriptions necessary to achieve steady-state growth and therefore to prepare an experimentally reproducible culture (for further discussion see p. 267–270 in reference 10).

Since the size and the composition of the E. coli cell are such sensitive functions of growth rate (chapter 97; see also discussion in chapter 6 of reference 10), it is recommended that the growth rate of a culture always be specified. The following items should be specified for any particular culture used to measure a parameter of interest: (i) the organism, identified by strain and source (e.g., "E. coli B/r, obtained from S. Cooper"); (ii) the medium (e.g., "glucose-morpholinepropanesulfonic acid [MOPS] minimal medium as described by . . ." [cite reference]); (iii) the aeration (e.g., "grown aerobically, with shaking [200 rpm, a 50-ml culture in a 250-ml Erlenmeyer flask with a Morton closure]"); (iv) the temperature (e.g., "37.5C"); (v) the growth phase (e.g., "balanced exponential growth, achieved by serial subcultivation through five mass doublings"); and (vi) the growth rate (e.g., "generation time = 42 min").

 

COMPOSITION OF AN AVERAGE E. COLI B/r CELL

The B and B/r strains of E. coli have been the subject of extensive biochemical and metabolic studies, more so than even the K-12 strains popular with geneticists. (Rapid growth in minimal medium, serving as host to T-even and other phages, and tight variance of cell division are some of the reasons that B and B/r strains have been favored by physiologists.) The information in Table 1 has been compiled from several sources, listed in the footnotes to the table. The compilation was guided by the article by Umbarger (17), and began with the overall percent composition data of Roberts et al. (13) for macromolecules. These data were then adjusted to match other information on glycogen (5), lipid (16), polyamines (9), and stable RNA/protein/DNA ratios (4). The dry weight per cell was determined in the author’s laboratory (F.C.N.) by weighing the dried cells removed by filtration from samples of a culture which had also been assayed for total cell content with the aid of an electronic particle counter. The calculated weight per average cell was found to be consistent with the size of the DNA genome and the number of copies of DNA predicted for a cell of average age. The water content was assumed from the value commonly cited in textbooks. This value has been recently confirmed in a very careful study (of a K-12 strain) that reported the water-accessible volume of the cell (divided between the cytosol and the periplasm) and the amounts of four significant osmolytes (K⁺ ion, glutamate, trehalose, and the organic buffer used in the medium) as a function of the osmolarity of the growth medium (3).

 

RESIDUE COMPOSITION OF E. COLI B/r PROTOPLASM

The information in Table 2 is derived heavily from the primary data of Roberts et al. (13) (cited in reference 17), though the amino acid analysis has been replaced by similar results from measurements in the author’s laboratory (F.C.N.).

The footnotes to the table present the sources of the other analytical data or assumptions used in the compilation or both.

 

FUTURE MOLECULAR INVENTORY

The tables in this chapter contain information useful for computing various parameters for E. coli. It is hoped, however, that the main function served will be to highlight the softness and incompleteness of the information and the need for a more detailed, as well as more accurate, inventory. Many powerful tools are now available to construct an accurate inventory. In the context of ongoing efforts to identify the individual genes and proteins of this organism (see chapters 84, 109, 110, 115, and 116), an explicit program to refine the overall chemical analysis of the cell makes great sense. Current interest in the assembly reactions of the cell, in the process of cell division, and in the operation of regulatory networks can benefit greatly from such an undertaking. As the time approaches for serious modeling of the E. coli cell, the absence of complete information on its chemical composition becomes strikingly apparent.