Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

Abstract

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F-Xwild-type strain, is shown here to carry a cryptic mutation, ftsRl, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42°C and the inability to grow in salt-free LB broth at 42°C.The ftsRl mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201(Mecr) and alaS21(Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a pc-reU' plasmid. These backgrounds also confer resistance in LB broth to the I-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsRI mutant (but not an isogenicftsR' strain) is sensitive to mecillinam in minimal glucose medium at 37°C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsRl mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37°C but not the growth defect in salt-free LB broth at 42°C. Genetic analysis indicated that the full phenotype of the ftsRl mutant is due to a single mutation in the rpoB gene (90 min), coding for the P subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.The cell cycle of Escherichia coli is tightly regulated. Septation normally begins when cells reach a precise length, suggesting the existence of a mechanism for coupling between cell wall elongation and septation. Such coupling mechanisms ensure a harmonious cell cycle. To understand them, one must identify the effectors and their targets, presumably elements of the septation apparatus. The isolation of conditional (fts) mutants that cannot divide at nonpermissive temperatures has been extensively used to define elements involved in E. coli cell division. This approach identified the essential gene ftsZ, whose product is required for an early step of septation and seems to be limiting for the process (45,47). This protein is a key control point of septation and the target of several endogenous cell division inhibitors (24). It has been shown that FtsZ, which is normally cytoplasmic, forms a ring around the cell center at the time of septation (3) and that this ring cannot be formed when the cell division inhibitor SfiA or MinCD is overexpressed (4). The FtsZ protein possesses a GTPase activity essential for septation; its GTP-binding site is homologous to those of eukaryotic tubulins, suggesting that the FtsZ protein may have a structural rather than an enzymatic function in septation (11,27,31).The peptidoglycan synthe...

Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli

“…A correlation between ppGpp accumulation and amdinocillin resistance has been previously demonstrated for E. coli (3,13,(26)(27)(28), and it has been of interest to determine whether this phenomenon is related to the ppGpp-dependent penicillin tolerance described here. Amdinocillin is a ␤-lactam agent which specifically inhibits penicillin-binding protein 2 (PBP 2).…”

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli

“…The lethality of mecillinam depends on the strain and on the culture medium. We have presented evidence that these variations reflect differences in the level and action of ppGpp Joseleau-Petit et al, 1994;Vinella and D'Ari, 1994).…”

“…We introduced the argS201 allele by co-transduction with eda::Tn10 ; the transductant was then transduced to eda þ (Tet S ) by selecting for growth on glucuronate. Other strains have been described: GC2553 (wild type), GC2702 (argS201 ) and GC3811 (GC2553/FЈlacI q /pGC401) (Joseleau-Petit et al, 1994), GC3782 (wild type) and GC3802 (argS201 ) (Vinella and D'Ari, 1994).…”

An Escherichia coli Mutant That Makes Exceptionally Long Cells

“…Minimal medium (pH 7.2) was prepared with 0.2% ammonium sulfate, 0.1 M potassium phosphate, 0.05% isoleucine, and 0.05% valine. Both LB and LBNS contained 1% tryptone and 0.5% yeast extract, and LB also included 0.5% sodium chloride (12). LBMin had the components of minimal medium plus 1% tryptone and 0.5% yeast extract.…”