Mutations in the ton (capR) gene result in multiple phenotypes, one of which is the failure to degrade abnormal and normal proteins (Deg-). Previous work with partially purified preparations showed that the Ion (capR) gene product is a 94,000-dalton polypeptide with an affinity for nucleic acids. The ton (capR) protein has now been highly purified and is demonstrated to have an ATP-dependent protease activity. The enzyme hydrolyzed 3H-labeled a-casein into trichloroacetic acid-soluble forms in Tris buffer containing Mg2+ and ATP. The reaction had a pH optimum of 8.5 and ATP was the preferred nucleotide. CTP Mutations in the ion (capR) locus ofEscherichia coli K-12 result in a host of phenotypic effects. These include the overproduction of capsular polysaccharide due to derepression of at least four spatially separated operons (1), failure to lysogenize A (2, 3) and P1 (4) phages, increased sensitivity to UV light and ionizing radiation (5,6), and filament formation and defective cell division (7,8). In addition, ion (capR) mutants exhibit reduced degradation ofnonsense (9-11), missense (12), and normal proteins (13). This abnormal proteolysis has been designated the Deg-phenotype (9, 10).Earlier work had implicated a requirement for energy in the breakdown of abnormal proteins in E. coli (14). Recently, energy inhibitors such as cyanide and azide have been shown to prevent the normal breakdown of a nonsense fragment of Pgalactosidase in vivo (11) and, in studies using cell extracts, the degradation of nonsense fragments was stimulated by the addition of ATP (15).

The polypeptide product of the Ion (capR) gene was identified and partially purified from bacterial strains homozygous for the capR' or capR9 (ochre mutation) alleles cloned with pSC1O1. A 94,000-dalton polypeptide was identified as the Ion (capR) gene product. Studies ofbinding to DNA cellulose columns and nitrocellulose filters indicate that the capR+ and capR9 proteins bind DNA.capR (ion) mutants of Escherichia coli K-12 are sensitive to UV light and ionizing radiation, and they overproduce capsular polysaccharide (colanic acid), as well as 10 enzymes involved in colanic acid synthesis (1). After irradiation, capR strains form nonseptate filaments that die (24). capR mutants of E. coli K-12 are very likely mutant in the same gene as is E. coli B (5), a radiation-sensitive strain ofbacteria discovered by Witkin (6). capR mutants also exhibit a reduced capacity to degrade abnormal (7-9) as well as normal (10) proteins. The use of in vitro cloning techniques permitted us to clone the capR+ (lon+ gene on an 8,200,000-dalton EcoRI DNA fragment (11,12). The capR+ plasmids (pBZ201 and pBZ203) specified two new polypeptides in minicells and maxicells (recA, uvrA, phr; ref. 13) having Mrs of 94,000 and 67,000 as determined by NaDodSOJ polyacrylamide gel electrophoresis. Plasmids containing recessive capR mutations were deficient in synthesis of94,000-dalton (dal)-polypeptide in maxicells, and a plasmid containing a dominant capR allele (capR9) overproduced a polypeptide with the same electrophoretic mobility as the 94-kDal one (12). These observations suggested that the 94-kDal species was the capR gene product that was defective in autoregulation in the strain containing the capR9 allele. However, in the absence of data showing that the capR9 form of the polypeptide was altered, another plausible interpretation is that the recessive and dominant mutations were in the capR gene that regulates synthesis of a second gene that specifies the 94-kDal polypeptide. In the present study, we partially purified the 94-kDal polypeptide from capR+ and capR9 homogenotes. Biochemical evidence is presented showing that the native form of the capR+-specified 94-kDal polypeptide is altered in the capR9 mutant and thus the capR (Ion) gene is the structural gene for it. The purified capR+ and capR9 proteins each bind to DNA with certain differences that are presented below.MATERLILS AND METHODS Buffers. Buffer A was 100 mM K2HPO4-KH2PO4, pH 6.5/ 10 mM 2-mercaptoethanol/1 mM EDTA/20% glycerol (vol/ vol). Buffer B was 10 mM Tris HCI, pH 7.1 at 25°C/1 mM 2-mercaptoethanol/1 mM EDTA/20% glycerol (voVvol) 20 mM NaCl. Buffer C was 20 mM Tris HCl, pH 7.5 at 25°C/50 mM NaCl/5 mM MgCl2/0. 1 mM EDTA/1 mM dithiothreitoV20% glycerol (vol/vol Growth of Bacteria and Preparation of Cell-Free Extracts. Bacteria were grown in complex medium at 370C to --2 X 108 bacteria per ml, isotope was added, and growth was continued to stationary phase. Bacteria were harvested, washed, and suspended in buffer A, disrupted by sonic oscillation, and centri...

The gene product of the pleiotropic lon (also called capR) locus in Escherichia coli, the CapR protein, is an ATP hydrolysis-dependent protease and a nonspecific nucleic acid-binding protein. We demonstrated that it is also a DNA-stimulated adenosine triphosphatase (ATPase). This new activity is distinct from the protease-associated ATPase activity and occurs in the absence of proteolytic substrate. The reaction requires the presence of a divalent cation and has a pH optimum of 8.0. The products of the reaction are ADP and inorganic phosphate. No adenylation or phosphorylation of the DNA or proteins was detected. The maximum rate of ATP hydrolysis occurs in the presence of supercoiled (form I) DNA. Relaxed circles (form II), double-stranded DNA, and single-stranded DNA are less effective in promoting ATPase activity, whereas RNA is inactive. The DNA-stimulated ATPase activity is inhibited by a mutationally altered form of the CapR protein called the CapR9 protein. The interaction of the CapR and CapR9 subunits suggests that this enzymatic activity of the CapR protein is oligomeric in the presence of DNA. Our in vitro experiments indicate a possible role for nucleic acids in the regulation of all lon (capR) activity.

The molecular biology of Mapuera virus was studied at both the protein and nucleic acid levels. Seven virusencoded proteins were detected in infected Vero cells. The sizes and characteristics of each of the proteins determined from various radiolabelling experiments allowed preliminary identification of the proteins as the large (L; 190 kDa), haemagglutinin neuraminidase (HN; 74 kDa), nucleocapsid (N; 66 kDa), fusion (Fo; 63 kDa), phosphoprotein (P; 49 kDa), matrix (M; 43 kDa) and non-structural (V; 35kDa) proteins. Western blot analysis showed that the HN, N and P proteins were major antigens recognized in the mouse. A cDNA library of total virus-infected cellular mRNA was created and screening of the library resulted in the detection of cDNA sequences representing the N mRNA transcript of Mapuera virus. The N mRNA sequence determined from the clones was 1731 nt in length and contained an ORF that encoded 537 amino acids, the complete 3' untranslated region and part of the 5' non-coding region. The calculated M r of the N protein was 59 kDa, which is close to the 66 kDa protein observed by SDS-PAGE.

Previous studies demonstrated that a cloned 2-megadalton (MDal) fragment of Escherichia coli DNA contained the structural gene for major outer membrane protein a (also known as 3b or M2 (40 kDal). The present study demonstrates that M2 is synthesized from a 42-kDal precursor that also is present in the outer membrane. The conversion of the 42-kDal precursor to M2 is inhibited by a number of different local anesthetics (procaine, piperocaine, lidocaine, cocaine), by the neuroactive drug atropine, and by the classical try in inhibitors Na-tosyllysine chloromethyl ketone (TLCK) and benzamidine. Our kinetic studies demonstrate that the amidase action of pure trypsin is inhibited competitively by the local anesthetics tested (excluding lidocaine) as well as by atropine and neostigmine. A mechanism of action for local anesthetics as well as atropine in E. coli may be to inhibit trypsinlike proteases, in a competitive manner, in the region of the outer membrane. The mechanism of action of these compounds in regulating nerve conduction in man may have certain features in common with the mechanism proposed in E. coli.One of the major outer membrane proteins of Escherchia coli K-12 is the 40-kilodalton (kDal) protein a (1, 2) [also known as 3b (3) and M2 (4); see ref. 5 for review]. Protein a is detected in the outer membrane of strains grown at 37°C but not at 30°C (2, 6). A 2-MDal cloned fragment of E. coli K-12 DNA contains the structural gene for protein M2 (4, 7), and studies with plasmid mutants demonstrated that this protein is important in repressing the synthesis of the capsular polysaccharide (8) in lon (capR) strains that overproduce the polysaccharide (unpublished data). The 2-MDal DNA fragment was originally isolated by using the cloning vehicle pSC101, the resultant plasmid being designated pMC44. Plasmid pMC44-coded proteins were determined by transforming a minicell-producing mutant to tetracycline resistance with the DNA, isolating the minicells, and incubating them in [-5S]methionine.Polypeptides were separated in sodium dodecyl sulfate (NaDodSO4) polyacrylamide gels by electrophoresis. A 42-kDal polypeptide, designated Ml, was also produced by plasmid pMC44-containing minicells. Several observations suggested that Ml is a precursor of the 40-kDal polypeptide M2: plasmid mutants were obtained that specified neither Ml nor M2 (unpublished data); the amount of Ml varied from 0 to 10% of the amount of M2; the amount of MI was influenced by the presence of tetracycline during [-5S]methionine-labeling as well as by the complexity of the medium in which the minicell-producing strain was grown (4). In addition, when plasmid pMC44 was in a minicell-producing strain that contained a Ion (capR) mutation that decreases proteolysis of nonsense and missense protein fragments (9-11), the amount of Ml detected by [35S]methionine-labeling of minicells was approximately equal to that of M2 (unpublished data).It is now established that precursor proteins exist for a number of bacterial proteins that are located ou...