The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12°C and 25°C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS^*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS^* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 M for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 M). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin

Summary Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Nov^r) mutants: 10% of Nov^r mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

Deletion of the prc (tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12

The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini. To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain. Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro. Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E. coli.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of -galactosidase with symmetric variants of - and -centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the -centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the -centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for -centered trp operator variants with exchanges in positions 3, 4 and 5.     

特异腐质霉角质酶-OMP25融合蛋白在大肠杆菌中的高效表达

【目的】通过探究特异腐质霉角质酶-OMP25融合蛋白(HiC-OMP25)在不同大肠杆菌(Escherichia coli)菌株中的表达情况、底物降解情况、热稳定性及宿主菌细胞膜通透性与细胞表面疏水性,揭示表达HiC-OMP25时不同宿主菌的差异性,并进一步提高HiC-OMP25在大肠杆菌中的表达量。【方法】分别在E.coli BL21(DE3)及E.coli C43(DE3)中表达HiC-OMP25,并测定其对对硝基苯丁酸酯(4-nitrophenol butyrate,pNPB)、聚丙烯酸乙酯(polyethyl acrylate,PEA)的降解效果、50℃稳定性;测定表达HiC-OMP25时宿主菌的细胞膜通透性及细胞表面疏水性变化;共表达伴侣蛋白提高HiC-OMP25在E.coli C43(DE3)中的表达量。【结果】HiC-OMP25在E.coli BL21(DE3)与E.coli C43(DE3)中均成功表达并降解pNPB,但前者对PEA的降解效果及50 ℃稳定性均低于后者。同时,表达HiC-OMP25显著增强了E.coli BL21(DE3)的细胞膜通透性及细胞表面疏水性。HiC-OMP25与巯基氧化酶(Erv1p)、二硫键异构酶(DsbC)在E.coli C43(DE3)中共表达时,其表达量为原始菌株的2.14倍,且对pNPB及PEA均有良好的降解效果。【结论】异源表达时,HiC-OMP25在E.coli C43(DE3)中正确折叠,而在E.coli BL21(DE3)中未完全正确折叠;通过共表达伴侣蛋白提高了HiC-OMP25在E.coli C43(DE3)中的表达量,为以后HiC-OMP25的工业化生产及应用奠定了基础。     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

Studies on the mechanism of reduction of W-inducible sulAp expression by recF overexpression in Escherichia coli K-12

UV-inducible sulAp expression, an indicator of the SOS response, is reduced by recF ⁺ overexpression in vivo. Different DNA-damaging agents and amounts of RecO and RecR were tested for their effects on this phenotype. It was found that recF ⁺ overexpression reduced sulAp expression after DNA damage by mitomycin C or nalidixic acid. recO ⁺ and recR ⁺ overexpression partially suppressed the reduction of UV-induced sulAp expression caused by recF ⁺ overexpression. The requirement for ATP binding to RecF to produce the phenotype was tested by genetically altering the putative phosphate binding cleft of recF in a way that should prevent the mutant recF protein from binding ATP that should prevent the mutant recF protein from binding ATP. It was found that a change of lysine to glutamine at codon 36 results in a mutant recF protein (RecF4115) that is unable to reduce UV-inducible sulAp expression when overproduced. It is inferred from these results that recF overexpression may reduce UV-inducible sulAp expression by a mechanism that is sensitive to the ability of RecF to bind ATP and to the levels of RecO and RecR (RecOR) in the cell, but not to the type of DNA damage per se. Models are explored that can explain how recF ⁺ overexpression reduces UV induction of sulAp and how RecOR overproduction might suppress this phenotype.     

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.