Growth-phase-dependent transcriptional regulation of the pcm and surE genes required for stationary-phase survival of Escherichia coli

Abstract

Two neighbouring genes, surE and pcm, a t 59 min on the Escherichia coli chromosome are both required for stationary-phase survival. Operon fusions of the putative promoter regions in front of surE (P2) or pcm (P3) with the lacZ reporter gene were constructed to study the transcriptional regulation of pcm and surE. Both promoter regions were able to activate pgalactosidase activity in a growth-phase-dependent way in either rich or minimal medium. Induction from both promoters reached the highest level in late stationary phase and was independent of the rpoSlkatF gene. Spent medium from early as well as late stationary-phase cultures could induce the expression of either promoter even after dialysis or boiling. A high cell density could induce the promoters more rapidly but not to a greater extent. It is proposed that the induction might be correlated with the decline in growth rate of the cells. The induction patterns of either P2 or P3 were very similar. pcm can thus be transcribed from both the P2 and P3 promoters that are regulated in similar ways.Keywords : operon fusion, stationary-phase survival, protein carboxyl methyltransferase INTRODUCTIONA protein-L-isoaspartyl 0-methyltransferase (EC 2.1.1.77) can specifically catalyse the transfer of the methyl groups from S-adenosylmethionine to atypical Lisoaspartyl but not normal L-aspartyl residues in proteins (Clarke, 1985). The wide distribution of the activity of this methyltransferase in the living world from eubacteria (Fu et al., 1991;Li & Clarke, 1992a) to fungi (Johnson et al., 1991), worms (Kagan & Clarke, 199S), plants (Mudgett & Clarke, 1993) and mammals (Ingrosso et al., 1989;O'Connor & Clarke, 1985), and the conservation of the amino acid sequence of the enzyme between species (e.g. 31 '/o identity between human and Escherichia coli) , indicate a house-keeping role of the enzyme (Fu et al., 1991 ;Ingrosso et al., 1989).Proteins or peptides with abnormal aspartyl residues derived from spontaneous protein degradation (Harding et al., 1989;Stadtman, 1988) are less reactive (Brennan et al., 1994; Johnson et al., 1987). Since methylation of the L-isoaspartyl residues facilitates the conversion of these residues to the normal form, it has been proposed that the methyltransferase might help to repair damaged proteins that can accumulate with time (Johnson et al., 1987;McFadden & Clarke, 1982. E. coli mutant strains without the pcrn gene (encoding protein carboxyl methyltransferase, Pcm) exhibit reduced stationaryphase survival and heat resistance (Li & Clarke, 1992b). These phenotypes are consistent with the hypothesis that the enzyme is involved in the metabolism of Lisoaspartyl-containing proteins that can accumulate.pcm is located at 59 min on the E. coli chromosome (Fu et al., 1991). Another gene, surE, that is also involved in the stationary-phase survival of E. coli is directly upstream of pcm (Li et al., 1994). Survival of Gramnegative bacteria in stationary-phase or nutrient-limited cultures that more closely resemble the conditions in the natu...

The RpoS Sigma Factor in the Dissimilatory Fe(III)-Reducing Bacterium Geobacter sulfurreducens

“…1). This is similar to E. coli, in which the transcription of surE, pcm, and rpoS is RpoS independent (10,20). The rpoS mutant DLCN16(⌬rpoS::Km) was constructed as described previously (18), using the oligonucleotides rpoS1 (5Ј-CTTACATGGTCGCCCTGATG-3Ј) and rpoS2 (5Ј-CATGGAGATCTCCGTCGC-3Ј) to amplify the upstream region of rpoS, oligonucleotides rpoS5 (5Ј-CGAGGCCAAGT CTCTGG-3Ј) and rpoS6 (5Ј-GCCGTATTCGAGCTGATAG G-3Ј) to amplify the rpoS downstream region, and oligonucleotides rpoS3 (5Ј-GCGACGGAGATCTCCATGACCTGGG ATGAATGTCAGCTAC-3Ј) and rpoS4 (5Ј-CCAGAGACTTG GCCTCGAGAAGGCGGCGGTGGAATCG-3Ј) to amplify the kanamycin cassette.…”

RpoS-dependent stress tolerance in Pseudomonas aeruginosa

“…stress than their exponential-phase parent strains (Small et al, 1994 (Li et al, 1997;Bohannon et al, 1991;Gordia & Gutierrez, 1996;Buchmeier et al, 1997 …”

“…rpoS gene homologues have been identified in other bacteria including Salmonella spp., Klebsiella pneumoniae, Shigella flexneri, Yersinia enterocolitica and Vibrio cholerae (Fang et al, 1992;Loewen & Hengge-Aronis, 1994;Small et al, 1994;Badger & Miller, 1995;Yildiz & Schoolnik, 1998). In these bacteria the RpoS homologues play a similar physiological role to that in E. coli.…”

Recovery from long-term stationary phase and stress survival in Escherichia coli require the l-isoaspartyl protein carboxyl methyltransferase at alkaline pH

“…Taken together, however, these results may suggest a new model for isoAsp protein repair in vivo. While PCM is active in exponential and early stationary phases (Li et al, 1997), its activity may be limited in long-term stationary phase, due to poor expression, instability, limiting concentrations of the methyl donor (S-adenosylmethionine) and/or excess of product (Sadenosylhomocysteine). If so, then even a repair-proficient cell would be unable to prevent accumulation of isoAsp damage during nutrient limitation, but it may be able to respond more rapidly to the return of favourable conditions when PCM is present to enable repair of existing proteins in addition to de novo synthesis.…”