PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63,000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65,000 68,000 and 72,000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510-amino-acid polypeptide had a calculated molecular mass of 55,426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a N-terminal segment of 30-amino-acid residues reminiscent of eukaryotic and prokaryotic signal peptides, and a hydrophobic domain of 21 residues near the C-terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface-layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze-etching electron microscopy performed on the wild-type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S-layer) which is in very close contact with other cell-wall components. These structures are absent from the cspB-disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the S-layer protein is the product of the cspB gene.

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The results presented in this paper establish relationships between structural, morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids.High-angle X-ray diffraction studies show that the order-disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases "cocrystallization" of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain.The various order-disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes.Discontinuities in the rate of D-lactate-dependent proline uptake as a function of temperature correlate with the order-disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate-dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains.The aim of the present work is to establish correlations between structural, morphological and physiological properties of membranes. Such studies have been undertaken previously and a variety of techniques have been used to determine the conformation of the paraffin chains of Escherichia coli membrane lipids, e.g. fluorescence [l, 21, electron spin resonance [3 -61 and high-angle X-ray diffraction [7,8]. The most direct technique is high-angle X-ray diffraction since it provides a quantitative evaluation of the fraction of the paraffin chains in the ordered and disordered conformation. Moreover, this technique allows an assessment of the segregation of different lipid species. Because of technical limitations however, high-angle X-ray diffraction has been used generally in a qualitative manner in the study of biological membranes. As a consequence, it has not yet been possible to determine the various lipid segregations taking place during the order-disorder transitions of the paraffin chains and to establish the correlations that exist between these segregations and the physiological properties of the membranes.Eur. J. Biochem. 49 (1974)

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