One of the major proteins of the Escherichia coli outer cell envelope membrane, protein II*, is shown to interact specifically with the same membrane's lipolysaccharide. In cell envelopes the protein (Mr∼ 33 000) is partially degraded by trypsin or pronase, and fragments with molecular weights of 24 000 or 19 000, respectively, are left with the outer membrane. The protein and these fragments in the cell envelope can act as receptors for a phage, and, most likely, as receptors in F‐mediated conjugation. Lipopolysaccharide protects isolated protein II* (in solution) in the same way against proteolytic attack, and the tryptic or pronase fragments are identical with those fragments obtained from protease‐digested cell envelopes. All fragments represent the N‐terminal moiety of the protein, and are, in the presence of lipopolysaccharide, active as phage receptors. Protein II* is heat‐modifiable in that its apparent molecular weight upon boiling in dodecylsulfate is 33 000 and without boiling is 28 000. Addition of lipopolysaccharide to the boiled species causes a renaturation to the species of molecular weight 28 000. Total E. coli phospholipid, synthetic dimyristoyl phosphatidylcholine, or the oligosaccharide moiety of lipopolysaccharide do not show any of these effects. However, the lipid part of lipopolysaccharide (lipid A) exhibits essentially the same activity as complete lipopolysaccharide. All results taken together strongly suggest that the interaction observed in vitro reflects such an interaction in vivo, and the possibility exists that this interaction is at least partially responsible for the specific cellular localization of the protein. Very likely a number of other major outer membrane proteins also interact specifically with lipopolysaccharide.

A procedure is described that from one batch of cells allows the isolation of all major proteins of the outer cell envelope membrane of Escherichiu coli B/r. The method involves differential extraction of cell envelopes with ionic and non-ionic detergents with and without Mg2+ present, and the proteins are finally separated by molecular sieve chromatography in the presence of sodium dodecylsulfate. From 200 g cell paste in ten days (including the five days chromatography) x 120 mg protein I (molecular weight x 38000), = 110 mg protein 11* (molecular weight = 33000), z 50 mg protein 111

Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pages, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.It remains unknown how proteins of the outer membrane of gram-negative bacteria are sorted to this membrane. We are studying this question with the 325-residue OmpA protein (7) of Escherichia coli (18,19,25,26) and have asked whether non-ompA mutants exist which might no longer incorporate the OmpA protein, and possibly others, into the outer membrane. Toward this end we have performed selections for resistance to an OmpA-specific phage under conditions which exclude the appearance of mutations in the ompA gene. Paradoxically, almost all mutants recovered so far possessed an unaltered OmpA protein, present in the outer membrane, and greatly reduced quantities of the outer membrane porins OmpC and OmpF, which have nothing to do with infection by this phage. Here we show that these mutants produced defective lipopolysaccharides (LPS). These and some other results shed some light on the role of LPS in the membrane assembly of these three proteins. MATERIALS AND METHODSBacterial strains, phages, and growth conditions. The ompA+ derivative of strain UH203 (19) is lac supF recA proA (or proB) rpsL(F' lacIq lacZAMJS proAB+). Its secA derivative, strain UH204, has the same characteristics but, in addition, is leu:: TnJO and secA(Ts5l) (17,44). The construction of a non mutant (49) in strain UH203 ompA+ was started with a precursor of this strain, JC6650 pro his (15). Phage P1 was grown on strain P400 (which is his' non [57]) and used to transduce the former strain to his'; the non allele is cotransduced at a frequency of -30% and was identified by the inability of strains carrying it to give rise to mutants resistant to phage T7,(49). The his+ non strain was made recA by t...

All 21 ribosomal proteins from 3 0 s subunits of Escheriehia coli were isolated in a pure state by the following methods: separation of subunits by zonal centrifugation in B XV rotors, extraction of proteins, CM-cellulose chromatography with pyridine formate gradients, gel filtration on Sephadex G-100 and, in some cases, preparative polyacrylamide block electrophoresis. The purity of isolated proteins was 97-100°/, shown by disc electrophoresis a t pH 4.5 in urea, by two-dimensional polyacrylamide electrophoresis, by dodecylsulfate gel electrophoresis and by cellulose acetate gel electrophoresis. The yield of the isolated proteins was 7 -110 mg depending on the protein and the number of purification steps.Fractionation of total 3 0 s proteins with ammonium sulfate is recommended for quick isolation of proteins S15 and 520 in large quantities.Waller [ 11 showed that starch gel electrophoresis separates the protein moiety of Escherichia eoli ribosomes into many bands. Later studies [2-101 demonstrated that the bands correspond to proteins of different physical and chemical structure. There exists also no immunological cross reaction between the individual 30 S proteins [ 111. There is agreement between various groups on the number, namely 21, and on the nomenclature of the ribosomal proteins from the 3 0 s subunits of E. coli [12].Good and reproducible methods for the isolation of ribosomal proteins on a big scale are the prerequisite not only for their chemical and physical characterization but also for protein-chemical studies on their amino sequences. The present paper describes t h e isolation of all 30 S ribosomal proteins of E. eoli in relatively large quantities (7-110 mg) and high purity (97-100°/,). Haan, Germany). After removal of cell debris and alcoa a t 16000 rev./min for 40 min in a Servall SS-34 rotor, ribosomes were sedimented by overnight centrifugation a t 25000rev.lmin in a Spinco 42 rotor. Ribosomes were resuspended in the buffer described above and purified by three cycles of differential centrifugation, the last with a buffer containing 0.5 M NH,C1 in 0.02 M Tris-HC1 pH 7.8,0.002 M MgC1, and 0.01 M mercaptoethanol. For our first isolations of ribosomes a buffer [14] containing 0.6 M (NH,),SO, instead of 0.5MNH4C1 was used for the last two cycles. MATERIAL AND METHODS IsolationFor separation of subunits aliquots of about 1.5 g ribosomes in 30 ml were dialyzed overnight against 5 1 of 0.01 M potassium phosphate pH 7.4. 0.001 hl MgCl, and 0.01 M mercaptoethanol and centrifuged in Spinco B XV Ti zonal rotors for 17 h a t 25000 rev./ min. A gradient of 7.5O/, to 38O/, sucrose in phosphate buffer and 600 ml overlay was used [15]. Absorbance was recorded a t 295 nm in a flow cell of a Beckman DB spectrophotometer. Subunits were precipitated either with ammonium sulfate (2/3 saturation a t 4 "C)

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