Low-temperature conditional cell division mutants of Escherichia coli

Abstract

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 were isolated. They grew normally at 390C but formed filaments at 300C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (390C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 250C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonuclease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division lesion in strain JS10 is cotransducible with maU, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome. ily applicable to "cold-sensitive mutants" (36). Thus, it seemed possible that the isolation and characterization of low-temperature conditional cell division mutants might reveal new and different loci involved in the cell division process. This paper descibes the physiology and genetic lesion in a UV-induced "cold-sensitive" mutant of E. coli K-12. MATERIALS AND MEIODS Bacterial strains. The bacterial strains and their genotypes are listed in Table 1. Genetic designations, map positions of amino acids, sugars, and antibiotics are in accordance with the revised linkage map of Bachmann et al. (4). Bacteriophage Plvir was part of the alive strain kit obtained from Cold Sprmg Harbor Laboratory, Cold Spring Harbor, N.Y. Strains JS20, JS101, and JS21 are maLA derivatives of strains x462, JS10, and LW3, repectively. The malA lesion was introduced into these three strains by 2-aminopurine mutagenesis (24) and subsequent selection of Mal7 colonies on MacConkey agar supplemented with 0.2% maltose. The glpD lesion was introduced into a X462 malA strain in an analagous fashion. Media. Dix and Helmatetter minimal medium (5) and Luria (L) broth (19) were used for isolation, enrichment, and subsequent experiments involving the low-temperature conditional cell division mutants.

Characterization of the ftsYEX operon of Escherichia coli

“…However, neither JS10 nor its parent strain was sensitive to other compounds known to challenge membranes, actinomycin D and lysozyme. JS10, however, was more sensitive than its parental strain to a number of antibiotics (47). Later analysis of revertants, i.e., no filamentation at the restrictive temperature, showed them to be as insensitive to antibiotics as the parent strain, while the sensitivity to deoxycholate persisted.…”

“…The state of the bacterial cell envelope was assessed by its sensitivity to deoxycholate. Five strains, including JS10, were unable to form colonies on L-agar plates containing 0.25% deoxycholate at either temperature (47). However, neither JS10 nor its parent strain was sensitive to other compounds known to challenge membranes, actinomycin D and lysozyme.…”

“…While previous temperature-sensitive mutants had been generated by chemical mutagenesis, these coldsensitive mutants were made by UV radiation and therefore were expected to reveal other loci important in cell division. These low-temperature mutants formed filaments at 30°C, but exhibited a "coordinated burst" of division forming short rods at the permissive temperature of 39°C (47). Nuclear staining revealed regularly spaced nucleoids throughout the filaments (grown at 30°C), which often contained one or two septa.…”

“…A paper published in 1978 by Sturgeon and Ingram reported the isolation of fifteen low-temperature conditional cell division mutants of E. coli (47). While previous temperature-sensitive mutants had been generated by chemical mutagenesis, these coldsensitive mutants were made by UV radiation and therefore were expected to reveal other loci important in cell division.…”

“…Nuclear staining revealed regularly spaced nucleoids throughout the filaments (grown at 30°C), which often contained one or two septa. Sucrose (10%) was effective in restoring growth to two of the CS mutants (47).…”