Mutations in sulA (sfiA) block the filamentation and death of capR (ion) mutants that occur after treatments that either damage DNA or inhibit DNA replication and thereby induce the SOS response. Previous suiA-iacZ gene fusion studies showed that sulA is transcriptionally regulated by the SOS response system (lexAlrecA). SulA protein has been hypothesized to be additionally regulated proteolytically through the capR (lon) protease, i.e., in lon mutants lacking a functional ATP-dependent protease there would be more SulA protein. A hypothesized function for SulA protein is an inhibitor of cell septation. To investigate aspects of this model, we attempted to construct Ion, Ion sulA, and Ion sulB strains containing multicopy plasmids specifying the suiA+ gene. Multicopy suIA+ plasmids could not be established in Ion strains because more SulA protein accumulates than in a Ion' strain. When the sulA gene was mutated by a mini-Mu transposon the plasmid could be established in the Ion strains. In contrast, suiA+ plasmids could be established in lon+, lon sulA, and Ion sulB strains. The suiA+ plasmids caused Ion sulA and Ion sulB cells to exist as filaments without SOS induction and to be sensitive to UV light and nitrofurantoin. Evidence implicated higher basal levels of SulA protein in these Ion plasmid suiA + strains as the cause of filamentation. We confirmed that the SulA protein is an 18-kilodalton polypeptide and demonstrated that it was induced by treatment with nalidixic acid. The SulA protein was rapidly degraded in a lon+ strain, but was comparatively more stable in vivo in a lon sulB mutant. Furthermore, the SulA protein was localized to the membrane by several techniques. enzymatic activities: an ATP hydrolysis-dependent protease activity (9, 10; M. F. Charette, Ph.D. thesis, University of Chicago, 1981), DNA stimulated ATPase activity (7a), and nucleic acid-binding activity (56; Charette, Ph.D. thesis). In addition, a defective CapR protein (capR9 allele) has also been purified. The CapR9 protein has retained the general nucleic acid affinity (9, 56), but has lost the protease activity (8,9). In Ion mutants, second site mutations in sul (suppressor of Ion) prevent the filamentation and UV sensitivity without affecting the mucoid phenotype of the cells (14,17,26,27). These mutants were isolated as UV-, nitro-NF-, or methyl methanesulfonate-resistant derivatives of lon strains. The sul mutations are located at two loci on the E. coli chromosome; sulA is near pyrD (22 min), and sulB is near leu (2 min). sfiA and sfiB (sfi for suppressor of filament induction) are identical to sulA and suiB, respectively, and were isolated as spontaneous thermoresistant revertants of tif-1 (recA441) Ion strains (15,23,24). The tif-l Ion strains filament and die at 41°C. To account for their data, George et al. (15) proposed that a division inhibitor (product of the sulA or sulB gene?) was induced by UV and that it might be more stable in Ion strains.By means of a Mu d(amp-lac) operon fusion that linked the structural gene for 3...
Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS‐polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.
A mutation in the Ion (capR) gene of Escherichia coli K-12 effects several phenotypic alterations in the mutant cell, such as overproduction of capsular polysaccharide and sensitivity to ultraviolet or ionizing radiation. A previously cloned 9.2-megadalton (Md) EcoRI fragment contained the capR+ gene and specified two polypeptides, 94 kilodaltons (K) and 67K. To provide evidence that the 94K polypeptide is the capR' gene product, we constructed a capR+ plasmid, pJMC40, having a 2.0-Md EcoRI-PstI fragment which codes only for the 94K polypeptide. Plasmids pJMC22 and pJMC30, having deletions of 0.7 and 0.8 Md, respectively, from one end of the 2.0-Md fragment, were also constructed. Each codes for a shortened stable polypeptide (from the 94K). Neither plasmid can
ftsQ is an essential cell division gene in Escherichia coli. The ftsQ gene has been sequenced, and a presumptive open reading frame has been identified; however, no protein product has been observed (A.C. Robinson, D.J. Kenan, G.F. Hatfull, N.F. Sullivan, R. Spiegelberg, and W.D. Donachie, J. Bacteriol. 160:546-555, 1984, and Q.M. Yi, S. Rockenbach, J.E. Ward, Jr., and J. Lutkenhaus, J. Mol. Biol. 184:399-412, 1985). The ftsQ gene was isolated on a 970-base-pair EcoRI-PvuII fragment of the E. coli chromosome and used to construct a trp-lac (Ptac) transcriptional fusion in plasmid pKK223-3. The fused construct (pDSC78) complemented an ftsQ1(Ts) mutant strain in trans, restoring growth at 42 degrees C on low-salt medium. An ftsQ1(Ts) mutant strain transformed with pDSC78 appeared normal upon microscopic examination, with no indication of filamentation. The ftsQ gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis of radiolabeled, isopropyl-beta-D-thiogalactopyranoside-induced maxicell and normal cell extracts. As predicted from the nucleotide sequence, the 970-base-pair EcoRI-PvuII fragment encoded a polypeptide of approximately 31,400 daltons. Analysis of the data obtained from pulse-chase experiments in maxicells and normal cells suggests that the FtsQ protein is stable. Most of the radiolabeled FtsQ protein from maxicells was found in the inner membrane. On the basis of available information, the prior inability to detect FtsQ can be attributed to low levels of transcription or translation rather than to proteolysis.
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