Process of Cellular Division in Escherichia coli : Physiological Study on Thermosensitive Mutants Defective in Cell Division

Abstract

Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described.While the genetic study of microorganisms has led to the description of a large number of important cell functions, the mechanisms of cell division and their regulation remain almost unknown.To analyze the regulatory mechanisms involved in cell division, a genetic approach was first taken. By the isolation and genetic characterization of conditional thermosensitive mutants of Escherichia coli defective in cell septation, seven genes (ftsA to ftsG) were identified (16,17,18,40; Ricard and Hirta, in press). Mutations in these genes cause the formation of multinucleated filaments.To gain insight into the mechanism of the septation process, a physiological study of new fts mutants was undertaken.

A class of gyrB mutants, substantially unaffected in DNA topology, suppresses the Escherichia coli K12 fts Z84 mutation

“…at 42"C/30''C of «1 x 10"^. Under the optical microscope, transductant celts, grown in NB (Ricard and Hirota, 1973), appear increased in size with respect to cells of the parental strain RS54 although they do not form real filaments. One of these clones, R308, was transformed with pUC13 and the plasmid DNA was analysed for degree of supercoiling after overnight growth at 3O''C both in LB (Miller, 1972) and NB medium.…”

“…Table 1 shows the efficiency of plating (e.o.p.) at various temperatures of the six clones both in the presence and the absence of NaCI in the growth medium (Ricard and Hirota, 1973). On the basis of the e.o.p., mutants can be grouped into two classes: one is able to suppress the ftsZoa with high efficiency (thermoresistant phenotype with an e.o.p.…”

“…LB (Miller, 1972) was used for standard cultures and plating. NB (Difco) (Ricard and Hirota. 1973) was used to analyse the effect of NaCl on the growth of R300 and its derivatives.…”

“…A number of genes of Escherichia C0//K12 involved in cell division have been identified on the basis of conditional lethal mutations which result in a filamenting, temperature-sensitive (fts) phenotype (Ricard and Hirota. 1973).…”

Escherichia coli Murein Transglycosylase

“…For the preparation of enzyme the Escherichia coli strain PA 3092 was used [4]. The cells were grown in nutrient broth (Difco) at 37 "C under aeration, supplemented with lysine (20 pg/ml), thymine (50 pg/ml), and meso-2,6-diaminopimelic acid (4 pg/ml), harvested at exponential growth phase (A578 0.7 -0.8) and stored at -70 "C until used.…”