Proteins induced in Escherichia coli by benzoic acid.

Proteins induced by benzoic acid in Escherichia coli were observed on two-dimensional electrophoretic gels (2-D gels). Cultures were grown in glucose-rich medium in the presence or absence of 20 mM benzoate at an external pH of 6.5, where the pH gradient (deltapH) is large and benzoate accumulates, and at an external pH of 8.0, where deltapH is inverted and little benzoate is taken up. Radiolabeled proteins were separated on 2-D gels and were identified on the basis of the index of VanBogelen and Neidhardt. In the absence of benzoic acid, little difference was seen between pH 6.5 and pH 8.0; this confirms that the mechanisms of protein homeostasis in this range are constitutive, including the transition between positive and inverted deltapH. Addition of benzoate at pH 6.5 increased the expression of 33 proteins. Twelve of the benzoate-induced proteins were induced at pH 8.0 as well, and nine of these matched proteins induced by the uncoupler dinitrophenol. Eighteen proteins were induced by benzoate only at pH 6.5, not at pH 8.0, and were not induced by dinitrophenol. One may be the iron and pH regulator Fur, which regulates acid tolerance in Salmonella spp. The other 13 proteins had not been identified previously. The proteins induced by benzoate only at a low pH may reflect responses to internal acidification or to accumulation of benzoate.     

Proteins induced by high osmotic pressure in Escherichia coli

Abstract The protein composition of Escherichia coli grown under conditions of high and low osmotic pressure and following shifts from one condition to the other was examined by two-dimensional gel electrophoresis. Upon shift to high osmotic pressure there is a rapid induction of a group of proteins whose synthesis persists during prolonged growth at elevated osmotic pressure. An osmotic down-shift results in the repression of these proteins. Neither shift induced the heat shock regulon.     

Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli

High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl–tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness.     

Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida, or Staphylococcus aureus.

Two-dimensional gel electrophoresis of proteins from Escherichia coli, Pseudomonas putida, and Staphylococcus aureus, grown with methionine or one of a variety of organosulfates and organosulfonates as the sole source of sulfur, showed expression of specific sets of 7 to 14 proteins which were not observed during growth with sulfate or cysteine for all three species or with thiocyanate for P. putida and S. aureus. Under the same conditions, arylsulfatase activity in P. putida and S. aureus was seen to increase by up to 140-fold, suggesting that the proteins induced under these conditions may be involved in sulfur metabolism. We propose that these proteins are members of a sulfate starvation-induced stimulon.     

Methylation of Ribosomal Proteins in Escherichia coli

Escherichia coli was grown in a medium containing [1-(14)C]methionine and [methyl-(3)H]methionine, and the (3)H/(14)C ratio was determined for each of the ribosomal proteins derived from the 70S ribosome. Evidence indicates that six proteins from the 50S subunit were methylated: L7, L9, L11, L12, L18, and L33. Methylation of several other 50S proteins (such as L1, L3, L5, etc.) may also occur. The methylated amino acids in protein L11 have been characterized further and found to be predominately epsilon-trimethyllysine. A small amount of a compound tentatively identified as N(G), N'(G)-dimethylarginine was also detected.     

Antibacterial activity of surfactants against Escherichia coli cells is influenced by carbon source and anaerobiosis

AIMS: In order to clarify the involvement of an energy-yielding system in the antibacterial action of surfactants, the effects of carbon source and anaerobiosis during the growth period on the surfactant sensitivity of Escherichia coli cells were investigated. METHODS AND RESULTS: Cetyltrimethylammonium bromide (CTAB) and N-dodecyl-N,N-dimethylglycine, at relatively low concentrations, caused a delay in growth of E. coli cells. Cells grown in M9 medium supplemented with glycerol, succinate or acetate as a carbon source were more sensitive to surfactants and had a higher respiratory activity than those grown with glucose. Cultivation under anaerobiosis made cells resistant to CTAB. CONCLUSIONS: Bacterial sensitivity to surfactants was affected by carbon source and anaerobiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained should be helpful in determining suitable conditions of treatment in the practical use of surfactants for bacterial decontamination.     

Enhanced Production of Recombinant Proteins in Escherichia coli by Filamentation Suppression

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.     

Proteins encoded by the Escherichia coli replication terminus region.

The replication terminus region (31 to 35 min) of the Escherichia coli chromosome contains very few mapped genes (two per min) compared with the remainder of the chromosome, and much of the DNA appears dispensable. In order to determine whether, despite this, the terminus region consists of protein-coding sequences, we cloned 44 kb (1 min) of terminus region DNA (that surrounding trg at 31.4 min) and examined its ability to catalyze protein synthesis in vitro or in minicells. We were able to account for more than half the coding capacity of the cloned DNA with proteins synthesized in these systems, indicating that the sparsity of mapped genes in the terminus region does not result from a lack of identifiable coding sequences. We can therefore conclude that the terminus region is composed mainly of expressable, albeit inessential, protein-encoding genes.     

Relative Stability of Membrane Proteins in Escherichia coli

The relative stability of membrane proteins in Escherichia coli was investigated to determine whether these proteins are degraded at heterogeneous rates and, if so, whether the degradative rates are correlated with the sizes or charges of the proteins. Cells growing in a glucose-limited chemostat with a generation time of 15 h were labeled with [¹⁴C]leucine. After allowing 24 h for turnover of ¹⁴C-labeled proteins, the cells were labeled for 15 min with [³H]leucine. By this protocol, the rapidly degraded proteins have a high ratio of ³H to ¹⁴C, whereas the stable proteins have a lower ratio. The total cell envelope fraction was collected by differential centrifugation, and the proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The relative ratio for each protein was determined by dividing its ³H/¹⁴C ratio by the ³H/¹⁴C ratio of the total membrane fraction. Although most of the 125 membrane proteins had relative ratios close to the average for the total membrane fraction, 19 varied significantly from this value. These differences were also observed when the order of addition of [¹⁴C]leucine and [³H]leucine was reversed. In control cultures labeled simultaneously with both isotopes, the relative ratios of these 19 proteins were similar to that of the total membrane fraction. Thirteen of these proteins had low relative ratios, which suggested that they were more stable than the average protein. An experiment in which the normal labeling procedure was followed by a 60-min chase period in the presence of excess unlabeled leucine suggested that the low relative ratios of 3 of these 13 proteins may be due to a slow post-translational modification step. Six membrane proteins had high relative ratios, which indicated that they were degraded rapidly. In contrast to the relationships found for soluble proteins in mammalian cells, there were no strong correlations between the degradative rates and either the isoelectric points or the molecular weights of membrane proteins in E. coli.     

Sensitivity of Escherichia coli to proline analogues during osmotic stress and anaerobiosis

L.M. REESE, K.O. CUTLER AND C.E. DEUTCH. 1996. The sensitivity of wild-type Escherichia coli K-12 to a series of proline analogues was determined in cultures containing increasing concentrations of NaCl under both aerobic and anaerobic conditions. The bacteria were most sensitive to L-azetidine-2–carboxylate and L-thiazolidine-4–carboxylate. The minimum inhibitory concentrations for these compounds decreased progressively during osmotic stress, but the bacteria were much more sensitive to these proline analogues under aerobic conditions than during anaerobiosis. The reduced sensitivity under anaerobic conditions did not reflect degradation of the compounds in the culture medium. Since both urine and medullary renal tissue contain relatively low oxygen concentrations, these results raise doubts about the potential use of proline or glycine betaine analogues in treating urinary tract infections.     

Gram positivity induced in Escherichia coli by chloroform-methanol treatment

Summary Extraction of lipid by chloroform-methanol from Escherichia coli induces Gram positivity in the cells of this bacterium. It has been observed that approximately 1.5 times more lipid is extracted from Escherichia coli than from Staphylococcus aureus by this treatment. An increase of 1.5–1.8 fold in the retention of dye has been evident in the case of S. aureus where as this was 70–90 fold in the case of E. coli, which is 1/4th of the normal retention by S. aureus. The more lipid present in the cell wall or cell membrane, the less retention of dye seems to be exhibited by the bacteria. This also substantiates our model for Gram reaction presented in a previous communication.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Proteins synthesized by a non-induced bacteriocinogenic factor in minicells of Escherichia coli

Summary The cloacinogenic factor Clo DF13 from Enterobacter cloacae has been transferred to the minicell-producing strain P678-54 of Escherichia coli K12. The data presented show that this Clo DF13 factor segregates into minicells of P678-54 (Clo DF13) and that this factor is the only plasmid, present in these minicells. Proteins from purified P678-54 (Clo DF13) and P678-54 minicells, previously labelled with ¹⁴C-amino acids, were compared after electrophoresis on SDS-polyacrylamide gels. From this comparison it appeared that a noninduced Clo DF13 factor directs the synthesis of 4 proteins. The molecular weights of these proteins could be estimated to be about 72000, 32000, 18500 and 12000. In P678-54 (Clo DF13) minicells, one additional Clo DF13 protein was found to be unlabelled. Apparently this protein is not synthesized in P678-54 (Clo DF13) minicells, but is segregated into or is attached to the minicells after being synthesized in the P678-54 (Clo DF13) cells. The molecular weight of this protein is about 62000, which corresponds to the molecular weight of cloacin.     

Effect of acetate on the growth of Escherichia coli during aerobiosis and anaerobiosis

The dynamics of acetate accumulation was studied in Escherichia coli K-12 batch cultures with a substrate addition. At pH 7.0, the growth stopped at 150 mmoles of acetate per litre of the medium under the aerobic conditions or at 35 mmoles of acetate per litre of the medium under the anaerobic conditions. Experiments with extraneous addition of acetate suggest that acetic acid plays a key role in inhibiting the growth of E. coli by acid metabolites. The authors propose a hypothetical mechanism to account for the inhibiting action of acetate.