Vol. 174, No. 16 JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5414-5423 0021-9193/92/165414-10$02.00/0 Copyright © 1992, American Society for Microbiology Isolation and Characterization of ftsZ Alleles That Affect Septal Morphology ERFEI BIt AND JOE LUTKENHAUS* Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66103 Received 10 March 1992/Accepted 11 June 1992 TheftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of theftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitiveftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery. The ftsZ gene was classified as a cell division gene on the basis of the filamentous phenotype conferred by the condi- tional lethal ftsZ84(Ts) mutation at the nonpermissive tem- perature (16). More recently this designation was confirmed when it was shown that the ftsZ gene was essential and that reducing expression of the gene led to inhibition of division (6, 18, 24). Surprisingly, however, the ftsZ84(Ts) mutation remains the only conditional lethal mutation in theftsZ gene, as no additional alleles were found among cell division mutants selected on the basis of a filamenting phenotype (9). In contrast, numerous conditional lethal mutations have been found in the ftsA and ftsI genes. In the absence of ftsZ function, observed in strains that conditionally express ftsZ or express the ftsZ84(Ts) muta- tion, cells form smooth filaments with no sign of constriction (6). Such morphology suggests that FtsZ acts earlier in cell division than other knownfts genes, since mutations in these genes result in filaments with indentations at regular inter- vals that may be aborted or stalled division attempts (9). In contrast to the effect of reducing ftsZ expression, slight overexpression offtsZ results in hyperdivision activity in the form of a minicell phenotype, although further overexpres- sion also inhibits division (1, 25). Such studies have sug- gested that FtsZ is rate limiting for division. Recent localization of FtsZ by using immunoelectron microscopy revealed that FtsZ is located at the leading edge of the septal invagination (4). The results demonstrated that FtsZ assembles at the midpoint of the cell in a ringlike structure prior to septation. This FtsZ ring is present throughout septation but disassembles upon completion of septation. It was suggested that the FtsZ ring forms a cytoskeletal element that determines the division site and activates the division process. A class of mutations, selected on the basis of resistance to the cell division inhibitor, SulA, map in the ftsZ gene and * Corresponding author. t Present address: Department of Biology, University of North Carolina, Chapel Hill, NC 27599. have been designated ftsZ(Rsa) (2). These mutations appear to alter the ftsZ gene product such that it is resistant to SulA (14, 15). These same mutations also confer increased resis- tance to the cell division inhibitor MinCD, which is part of the min system (3). Although these mutations are not con- ditional lethals, at least some of these ftsZ(Rsa) mutations confer a slightly temperature-sensitive cell division pheno- type, especially at low salt concentrations (10, 12, 13). Most of these ftsZ(Rsa) mutations were isolated at the chromo- somal locus; however, ftsZl(Rsa), ftsZ2(Rsa), and ftsZ3 (Rsa) were obtained on a low-copy vector in the presence of a wild-type allele on the chromosome (2). Of these alleles it was demonstrated that ftsZ3(Rsa) conferred resistance to SulA and MinCD in the presence of ftsZ but could not substitute forftsZ for cell viability. Further examination of the properties of ftsZ3(Rsa) indicated that FtsZ must func- tion as a multimer. In this paper we further characterize the ftsZ2(Rsa) muta- tion, and during this process we isolated additional condi- tional lethal mutations in the ftsZ gene. Each of these mutations has dramatic effects on cell physiology, including altered septal morphology, minicell formation, and cell lysis. MATERIALS AND METHODS Bacterial strains, phages, and plasmids. The strains used are all derivatives of MC4100 and W3110 and are listed in Table 1. Phage X 16-2 (imm21) is a transducing phage carrying a 10-kb chromosomal insert including the ftsZ gene (16). X BEF2 is identical to X 16-2 except that it carries the ftsZ2(Rsa) mutation (2). XDB173 carries the minCD genes downstream of the lac promoter and has been described elsewhere (8). pKD3 contains the ftsA, ftsZ, envA, and gene X genes on a BamHI fragment in the temperature-sensitive pSC101 derivative pEL3 (6). pBEFO contains the same BamHI fragment in pGB2, also a derivative of pSC101 (1). pBEF2 is identical to pBEFO except that it contains ftsZ2(Rsa) (2). pBEF20 contains the recA gene cloned into a pBR322 derivative that is Kanr. pELEZ2 contains the 5' truncatedftsZ gene on an EcoRI fragment cloned into pEL3. 5414