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...

capR(lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing." Furthermore, capR mutants are poorly lysogenized by bacteriophage X. Secondsite revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, suiB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+I pyrD36 sulAl 7 capR9 (Ion) demonstrated that sulA + is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation: complex medium-induced killing and the inability of X phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (Ion) and sul gene 1208 on August 1, 2020 by guest

A plasmid, pDLL4, was isolated from a Tn5tacl mutagenesis experiment with plasmid pZAQ. When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted). The TnStacl transposon was integrated within the carboxyl end of thejfsA gene in pDLL4. This mutation was designatedft&As. SubcloningJts&A DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene. DNA sequence analysis of the Jft4C mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added. A radioactive peptide band corresponding to this truncated FtC protein was identified by a T7 promoter-T7 polymerase protein labeling system. Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm. The ends of these aggregates appear to be at or near the cell membrane. The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm.

DNA fragments encoding the ftsA gene were subcloned into plasmids downstream of a lac promoter or a tac promoter. These plasmid constructs, when transformed into wild-type and mutant strains, inhibited normal cell septation, causing the formation of long nonseptate filaments. This phenotype is due to overproduction of the FtsA protein.

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.