Examination of the Tn5 transposase overproduction phenotype in Escherichia coli and localization of a suppressor of transposase overproduction killing that is an allele of rpoH

Abstract

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Tnp overproduction causes cell filamentation, abnormal chromosome segregation, and an increase in anucleated cell formation. There are two simple explanations for the observed phenotype: induction of the SOS response or of the heat shock response. The data presented here show that overproduction of Tnp neither induces an SOS response nor a strong heat shock response. However, our experiments do indicate that induction of some 32 -programmed function(s) (either due to an rpoH mutation, a deletion of dnaK, or overproduction of 32 ) suppresses Tnp overproduction killing. This effect is not due to overproduction of DnaK, DnaJ, or GroELS. In addition, Tnp but not ⌬11 Tnp (whose overproduction does not kill the host cells) associates with the inner cell membrane, suggesting a possible correlation between cell killing and Tnp membrane association. These observations will be discussed in the context of a model proposing that Tnp overproduction titrates an essential host factor(s) involved in an early cell division step and/or chromosome segregation.Tn5 is a composite 5.8-kb transposon that contains 1.5-kb inverted repeats IS50R and IS50L (see Transposition is a very rare event and responds in a complex fashion to the presence of various proteins (4). For example, the frequency of Tn5 transposition is dependent on the ratio between the two proteins encoded by the element itself, Tnp and Inh (61), and is influenced by a variety of host factors (3, 44), such as integration host factor (32), HU (32), DNA polymerase I (49), DnaA (3, 44), topoisomerase I (52), DNA gyrase (24), Dam methylase (64), Fis (58), and SulA (50, 57). The observation that Dam methylase regulates both the synthesis of Tnp and the transposition event itself suggests that transposition preferentially takes place soon after the DNA replication fork passes when the donor DNA is hemimethylated (44,63). This would allow the use of the sister chromosome to fill in the gap left by the excision of Tn5. Thus, Dam DNA methylation probably regulates the timing of the transposition event. This model was also described for Tn10 (4). The mechanism by which other host factors influence the frequency of Tn5 transposition is not yet clear. Moreover, there could be other unknown host factors that affect Tn5 transposition.Overproduction of Tn5 Tnp is lethal to Escherichia coli in the absence of Tn5 end sequences (therefore, in the absence of transposition). Overproduction killing is associated with both filament formation and defective nucleoid segregation. This Tnp overproduction killing has been partially characterized (59). Through an analysis of various Tnp mutants, it has been determined that catalytically active transposase is not required for killing. However, analysis of N-terminal deletions of Tnp showed that deletion of the first 3 or 11 amino acids from the N terminus blocked the killing. The wild-type N terminus of Tnp therefore must be very critical for the cell killing phenotype, possibly due...

Induction of the SOS response in starved Escherichia coli

“…Observations were made according to Yigit and Reznikoff [1997]. Briefly, bacteria were collected and resuspended in sterile saline (0.9% NaCl in H 2 O), spread onto glass slides, then air-dried and fixed with 100% methanol.…”

Direct Interaction between Escherichia coli RNA Polymerase and the Zinc Ribbon Domains of DNA Topoisomerase I

“…RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10). The proposed function of topoisomerase I activity in removal of transcription-driven hypernegative supercoiling (8,9) would be greatly facilitated by direct proteinprotein interaction with RNA polymerase.…”

“…In previous studies, Tn5 transposase was found to copurify with E. coli DNA topoisomerase I and inhibit the topoisomerase I activity (10). RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10).…”

SeqA Protein Stimulates the Relaxing and Decatenating Activities of Topoisomerase IV

“…Overexpressed proteins were purified as described previously (26,36,37). Subunits of gyrase and topo IV were mixed at equimolar concentrations then incubated on ice for 15 min to allow for holoenzyme assembly before assaying.…”