Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon

Abstract

The mini-TnlO transposon (A16A17TnJO) confers tetracycline resistance. When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene. Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes. If the mini-TnlO inserts between an essential bacterial gene and its promoter, th,e insertion mutation can cause conditional growth which is dependent on the presence of tetracycline. Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants. These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon. The pdxJ operon has not been described previously. It consists of two genes, pdxJ and dpj. Whereas the dpj gene is essential for E. coli growth in all media tested, pdxJ is not essential. The pdxJ gene encodes a protein required in the biosynthesis of pyridoxine (vitamin B6).Conditional mutations facilitate the study of bacterial genes in two ways. They allow detection of physiological changes under nonpermissive conditions and provide the opportunity to select suppressor mutations. In addition to conditional lethal mutations of the thermosensitive type, conditional detects dependent on expression from heterologous promoters are also valuable for study. Chow and Berg (6) designed a transposition vector, TnStacl, to detect essential genes in Escherichia coli as conditionally lethal transposition mutations. Mutants generated depend on induction of the tac promoter from the transposon to express essential genes when their normal expression has been blocked by insertion of the transposon. We show here that a TnJO derivative can also be used to isolate a class of conditionally lethal mutations.We have defined three genes of the mc operon: mc, era, and recO (31). These genes encode the proteins RNaseIII, a double-strand-dependent endoribonuclease (7); Era, a GTPbinding protein (5, 21); and RecO, required for DNA repair and RecF pathway recombination (25). Of these, only era is essential for bacterial growth (14, 31). We previously isolated an E. coli mutant in which a mini-TnlO (A16A17TnJO; 33) inserted in the leader sequence of the mc operon caused the cell to become conditionally dependent on tetracycline for growth. This insertion mutation, mc-40, caused era expression to be defective in the absence of tetracycline presumably because the mini-TnlO element was blocking normal transcription from the mc promoter. We proposed that tetracycline, which induces the divergent promoters for the tetA and tetR genes of the mini-TnlO transposon (13), allowed transcription of these genes to continue beyond the TnlO element and into the era gene. In this study, we have examined expression of lacZ fused to the mc operon both with and without the mc-40 mini-TnJO insertion (A40). We have also isolated and ...

Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

“…The results of this study show that the AEra protein exhibited a reduced GTP-hydrolysis activity and an increased GTP-binding activity, and failed to bind to the E. coli cytoplasmic membrane. The truncated era (Aera) gene was unable to complement an E. coli mutant defective in Era production (Takiff et al, 1989(Takiff et al, , 1992. Together, these results suggest that the Cterminus of the Era protein is essential for the in viuo function of the protein.…”

“…HT120 contains a mini-Tnl0 insertion upstream of the rnc gene which forms part of an operon with its downstream gene, era (Takiff et af., 1989(Takiff et af., , 1992. The mini-Tn 2 0 carries two divergent Tet-inducible promoters (Takiff et al, 1989(Takiff et al, , 1992. In the absence of Tet, HT120 could not grow on LB plates because the mini-Tnl0 insertion is downstream polar to era, which in turn blocks the transcription of the era gene.…”

“…defective in Era production, HT120 (Takiff et al, 1989(Takiff et al, , 1992. HT120 contains a mini-Tnl0 insertion upstream of the rnc gene which forms part of an operon with its downstream gene, era (Takiff et af., 1989(Takiff et af., , 1992.…”

“…Recently, a human homologue of Era was reported that is distincr from RAS proteins (Britton et al, 1998 The GenBank accession number for the nucleotide sequence of the Streptococcu:: pneumoniae era gene is AF07281. negative bacteria, and Mycopfasma spp. (Ahnn et al, 1986;Fleischmann et al, 1995;Fraser et al, 1995;Kawabata et al, 1997;Zuber et al, 1990Zuber et al, , 1997, and is essential for bacterial growth (Gollop & March, 1991b;Inada et al, 1989;March et al, 1988;Sat0 et al, 1998;Takiff et al, 1989Takiff et al, , 1992. Era proteins are highly conserved among different bacteria and appear to share the same function as evidenced by the fact that era genes from quite diverse bacterial species are able to complement E. coli era mutants (Pillutla et al, 1995;Zuber et af., 1990Zuber et af., , 1997.…”

Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl Transfer in Escherichia coli

“…Takiff et al (10) first identified the E. coli open reading frame containing the gene that encodes AcpS. At the time, this gene was of unknown function and was named dpj.…”