The synthesis of the Escherichia coli capsular polysaccharide varies with growth medium, temperature of growth, and genetic background. lac fusions to genes necessary for capsule synthesis (cps) demonstrated that these genes are regulated negatively in vivo by the lon gene product. We have now isolated, characterized, and mapped mutations in three new regulatory genes (rcs, for regulator of capsule synthesis) that control expression of these same fusions. rcsA and rcsB are positive regulators of capsule synthesis. rcsA is located at min 43 on the E. coli map, whereas rcsB lies at 47 min. rcsC, a negative regulator of capsule synthesis, is located at min 47, close to rcsB. All three regulatory mutations are unlinked to either the structural genes cpsA-F or lon. Mutations in all three rcs genes are recessive to the wild type. We postulate that lon may regulate capsule synthesis indirectly, by regulating the availability of one of the positive regulators.

Celis containing the pleiotropic Escherichia coli mutation ion filament extensively and die after exposure to ultraviolet light. Outside suppressors of the ultraviolet sensitivity, called sul, have previously been described at two loci; these mutations reverse the ultraviolet sensitivity of Ion strains but do not affect the mucoidal or degradation defect of these strains. An isogenic set of strains carrying combinations of Ion, suA, and sulB was constructed, and their behavior during normal growth and after ultraviolet treatment was studied. suA mutations had no detectable phenotype in Ion+' cells; the Ion suA strains filamented transiently after ultraviolet irradiation, as did lon' sul' cells. We found that the sulB mutation, which alters cell morphology and slows recovery from transient filamentation after ultraviolet treatment, was epistatic to both Ion and suA. Whereas sulA mutations were recessive to the wild-type allele, sulB was partially dominant. The simplest model to account for our observations is that sulA and Ion participate in a pathway of filamentation independent of that which produces transient filamentation in wild-type strains; sulB product may be the target of suA action and may play a role in normal cell division.

Certain Escherichia coli rho mutations, exemplified by rhoO26, block the growth of phage A by interfering with phage gene expression. The phage gene N, whose product suppresses transcription termination, appears to be expressed normally in the mutants, and the functional stability of the N protein is not affected. Our data suggest that these rho mutations allow transcription to terminate despite the presence of N. Other E. coli mutants displaying a similar phenotype (Nus-) fail to propagate wild-type A but permit the growth of the A variant Anin5, which has undergone a deletion of the A terminator tR2. The phenotype of the rhoO26 mutant differs: the growth of A is only marginally improved by the nin5 deletion. Interestingly, N activity at rho-independent terminators is not inhibited by the mutations, whereas its ability to suppress rho-dependent terminators is markedly reduced. The relevance of this specificity in terms of models of N action is discussed.

It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A. Markovitz, in I. Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977). To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M. J. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S. A. 76:4530-4533, 1979). At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains. These genes are located in a cluster close to udk at 45 min on the E. coli map; we have named these genes cpsA, B, C, D, and E. An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis. Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus. Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway. 184 on August 3, 2020 by guest

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.