Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.     

Studies on the endogenous metabolism of Escherichia coli

1. The endogenous metabolism of Escherichia coli has been studied by examining changes in cellular composition and of the suspending fluid during starvation of washed suspensions of the organism, in water or in phosphate buffer, at 37° under aerobic and anaerobic conditions. 2. When E. coli is grown in glucose–ammonium salts media the cells contain glycogen, which is utilized rapidly during subsequent starvation of the cells. 3. Ammonia is released by starved cells only after a lag period, which corresponds to the time taken for the cellular glycogen to be almost completely utilized. 4. If cells are grown under conditions that permit incorporation of ¹⁴C into protein but not into glycogen and are then starved, release of ¹⁴CO₂ commences immediately and continues at a linear rate throughout the period of glycogen utilization; it is concluded that the presence of glycogen in the cell prevents the net degradation of nitrogenous materials but does not suppress protein turnover. 5. RNA is degraded by the cells immediately they are starved, ribose is oxidized and ultraviolet-absorbing materials are released to the suspending medium. 6. There is no significant utilization of lipid during the starvation of glucose-grown E. coli. 7. There is no loss of viability during the initial 12hr. period of starvation under either aerobic or anaerobic conditions, but thereafter the cells die more rapidly under conditions of anaerobiosis. 8. These results are discussed in relation to the known patterns of endogenous metabolism and survival of other bacteria.     

Relation of Lipopolysaccharide and Fatty Acid Ester Release to the Ethylenediaminetetraacetic Acid Alteration of Permeability in Enterobacteriaceae

Escherichia coli subjected to cold osmotic shock released 30 to 40% of their fatty acid esters and 42% of their cellular hexosamine. In contrast, Enterobacter, although they released 40% of fatty acid esters, release only 25% of hexosamine. Proteus released less than 15% of either fatty acid esters or hexosamine. These differences are taken to explain the differences among the Enterobacteriaceae in releasing surface enzymes after osmotic shock. It is felt that the release of additional lipopolysaccharide after osmotic shock is necessary for the release of surface enzymes that are not freed by ethylenediaminetetraacetic acid-tris(hydroxymethyl)aminomethane exposure.     

Cell Division of Escherichia coli: Control by Membrane Organization

Cells of certain strains of Escherichia coli, after transfer from 37 to 45 C and incubation for 16 min, were observed to swell and subsequently divide synchronously. This swelling and the resulting stretching of the membrane are proposed to be the basis for the synchronous division. Four lines of evidence support this hypothesis. First, osmotic protection by the addition of either sodium chloride or sucrose at the time of heat shock prevents both swelling and synchrony. Second, a mutant neither swelled nor divided synchronously after heat shock. Third, cells grown for several generations with 10% sucrose in the medium swelled and divided synchronously upon transfer to medium without sucrose. Fourth, the mutant not synchronized by heat shock also swelled and underwent synchronous division after the osmotic shift. A tentative model is suggested for the normal control of division, based on membrane configuration at the septation site.     

Effects of amino acids on polysaccharide synthesis ofEscherichia coli K-12lon + andlon − strains

Ultraviolet-sensitivelon ^? mutant ofEscherichia coli K-12 produced abundant polysaccharide when grown in a minimal medium at 37 C, but not when grown in a broth medium. The repression of polysaccharide synthesis in the broth-grownlon ^? andlon ⁺ cells was studied. The effects were largely dependent on the amino acid concentrations and on the requirements of the strain used. At 200 μg per ml of each of the essential amino acids, histidine, proline, and threonine, there was complete inhibition of polysaccharide synthesis. At 200 μg per ml the required amino acids, tryptophane and tyrosine promoted polysaccharide synthesis. Most amino acids inhibited cell growth at 200 μg per ml but the inhibiting effect was smaller at 400 μg per ml. Polysaccharide synthesis of cells was not correlated with the growth rate, and occurred even under non-growing conditions.     

Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone

Experiments were performed under several conditions seeking evidence for turnover of protein-bound lipoic acid in Escherichia coli analogous to that described for the 4′-phosphopantetheine moiety of the E. coli acyl carrier protein. Pulse-chase, chase experiments using both low and saturating concentrations of lipoic acid, chase experiments in the presence of chloramphenicol, which prevents incorporation of lipoic acid into the protein-bound form, and chase experiments in cells in which the free pool of lipoic acid was reduced by osmotic shock all failed to demonstrate any turnover of protein-bound lipoic acid.     

Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.     

Stabilization of Homoserine-O-Succinyltransferase (MetA) Decreases the Frequency of Persisters in Escherichia coli under Stressful Conditions

Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in Escherichia coli cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-o-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in E. coli growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native metA gene on the E. coli K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in E. coli K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.     

Transient,specific and extremely rapid release of osmolytes from growing cells of Escherichia coli K-12 exposed to hypoosmotic shock

The influence of hypoosmotic shock on the solute content of growing Escherichia coli K-12 cells was investigated at 37°C. Within 20 s after the shock the cells had released most of their osmolytes K⁺, glutamate and trehalose. This release was specific and not due to rupture of the cell membrane, since under these conditions i) the cells neither lost protein nor ATP, ii)[¹⁴C]-labeled sucrose did not enter the cytoplasm from the periplasm, and iii) except for their glutamate and aspartate level, which decreased, the amino acid pool of alanine, lysine and arginine of the cells remained approximately constant. Within a minute after the shock the cells started to reaccumulate parts of their previously released glutamate, aspartate and K⁺, but not trehalose and resumed growth within 10 min after the shock. Experiments with K⁺-transport mutants showed that none of the genetically-identified K⁺ transport systems is involved in the K⁺-release process. Reaccumulation of K⁺ took place via the uptake systems TrkG and TrkH. The possibility is discussed that the exit of solutes after hypoosmotic shock occurs via several stretch-activated channels, which each allow the release of a specific osmolyte.Abbreviations OD₅₇₈ optical density at 578 nm - TEA triethylammonium - TMG 1,-S-methyl--thiogalactopyranoside     

Osmotic Behavior of Bacterial Protoplasts: Temperature Effects

Among protoplasts released from cells of Bacillus megaterium grown at 20, 30, or 37 C, osmotic swelling in NaCl solution at a given external osmotic pressure was greatest for protoplasts from cells grown at 20 C and least for protoplasts from cells grown at 37 C. Protoplasts from cells grown at lower temperaturs were also less stable to osmotic shock and lysed at higher external osmotic pressures than did protoplasts from cells grown at higher temperatures. But for cells grown at any one temperature, osmotic stabilization was itself temperature dependent so that the higher the ambient incubation temperature, the higher the osmotic pressure needed to prevent lysis of a given fraction of the input protoplast population. However, comparison of the osmotic stability of protoplasts from cells grown at different temperatures at various ambient incubation temperatures revealed that, except at 5 C where no differences were discerned, protoplasts from cells grown at lower temperatures still lysed at higher osmotic pressures than did those from cells grown at higher temperatures. The apparent internal osmolality (28 to 31 atm) did not vary significantly among whole cells from the three growth temperatures. Therefore, the observed differences in osmotic behavior could not be attributed to changes in internal osmotic pressure. Rather, it seemed likely that the differences were due to changes in membrane properties.     

Replication of plasmid chimera pBR-mtB-A containing rat liver mtDNA fragment in Escherichia coli polA cells

Escherichia coli K-12 strains p108 (polA6), p3478 (polA1), and KS55 (polA12, ts) deficient in DNA polymerase I were transformed by recombinant pBR-mtB-A plasmid containing BamHI-A fragment of rat liver mtDNA and pBR322 plasmid. The physical map of the pBR-mtB-A, containing the recognition sites for SalI, EcoRI and HinIII endonucleases, was constructed and the orientation of mtDNA fragment joined to pBR322 plasmid was studied. The phenotypic selection using ampicillin containing medium at permissive and nonpermissive temperature (KS55 strain), or at 37 °C (polA6 and polA1 strains) revealed that only the cells transformed with the hybrid plasmid are able to grow under these conditions. The presence of mtDNA insertions in chimeric DNA molecules of pBR-mtB-A in polA strains was proved by electrophoretic and hybridization analysis. Thus the results obtained demonstrate the replication of the vehicle containing both plasmid replicon and mitochondrial origin in the conditions nonpermissive for the stable reproduction of the plasmid DNA alone.     

The release of endonuclease I from Escherichia coli by a new cold shock procedure

A new cold shock procedure has been developed for releasing large quantities of endonuclease I from $\text{E. coli}$, which neither involves EDTA-lysozyme treatment nor osmotic shock. Treatment of cells with ice-cold 0.1M Tris-0.2M KCl buffer, pH 7.4 results in the release of endonuclease I into the medium. Although the loss of endonuclease I from the cells is a rapid process, its recovery in the shock fluid is gradual and approaches maximum in about 90 minutes. Certain divalent metal ions such as Mg⁺⁺ and Mn⁺⁺ strongly inhibit the release of endonuclease I. The cold shock procedure is rather selective and the mechanism of the release of endonuclease I is different from that of osmotic shock procedure.     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

Heat-Induced Expression and Chemically Induced Expression of the Escherichia coli Stress Protein HtpG Are Affected by the Growth Environment

Differences in expression of the Escherichia coli stress protein HtpG were found following exposure of exponentially growing cells to heat or chemical shock when cells were grown under different environmental conditions. With an htpG::lacZ reporter system, htpG expression increased in cells grown in a complex medium (Luria-Bertani [LB] broth) following a temperature shock at 45°C. In contrast, no HtpG overexpression was detected in cells grown in a glucose minimal medium, despite a decrease in the growth rate. Similarly, in pyruvate-grown cells there was no heat shock induction of HtpG expression, eliminating the possibility that repression of HtpG in glucose-grown E. coli was due to catabolite repression. When 5 mM phenol was used as a chemical stress agent for cells growing in LB broth, expression of HtpG increased. However, when LB-grown cells were subjected to stress with 10 mM phenol and when both 5 and 10 mM phenol were added to glucose-grown cultures, repression of htpG expression was observed. 2-Chlorophenol stress resulted in overexpression of HtpG when cells were grown in complex medium but repression of HtpG synthesis when cells were grown in glucose. No induction of htpG expression was seen with 2,4-dichlorophenol in cells grown with either complex medium or glucose. The results suggest that, when a large pool of amino acids and proteins is available, as in complex medium, a much stronger stress response is observed. In contrast, when cells are grown in a simple glucose mineral medium, htpG expression either is unaffected or is even repressed by imposition of a stress condition. The results demonstrate the importance of considering differences in growth environment in order to better understand the nature of the response to an imposed stress condition.     

Novel Insights into E. coli’s Hexuronate Metabolism: KduI Facilitates the Conversion of Galacturonate and Glucuronate under Osmotic Stress Conditions

Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal E. coli of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized E. coli proteins KduD and KduI in vitro. Galacturonate and glucuronate induced kduD and kduI gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In E. coli, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, kduID-deficient E. coli had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type E. coli. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in E. coli and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes.     

Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin–SUMO dual fusion system

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli.     

The anaerobic oxidation of dihydroorotate by Escherichia coli K-12

The oxidation of dihydroorotate under anaerobic conditions has been examined using various mutant strains of Escherichia coli K-12. This oxidation in cells grown anaerobically in a glucose minimal medium is linked via menaquinone to the fumarate reductase enzyme coded for by the frd gene and is independent of the cytochromes. The same dihydroorotate dehydrogenase protein functions in both the anaerobic and aerobic oxidation of dihydroorotate. Ferricyanide can act as an artificial electron acceptor for dihydroorotate dehydrogenase and the dihydroorotate-menaquinone-ferricyanide reductase activity can be solubilised by 2 M guanidine · HCl with little loss of activity.     

Sodium-Stimulated Glutamate Transport in Osmotically Shocked Cells and Membrane Vesicles of Escherichia coli

Three phenotypically distinct strains of Escherichia coli B were studied: one in which the transport of glutamate was strongly stimulated by sodium, one in which the transport was relatively independent of sodium, and one which did not transport glutamate. Membrane vesicle preparations from the three strains followed the behavior of whole cells with respect to sodium-stimulated transport. Although glutamate-binding material could be released from cells by osmotic shock, its affinity for glutamate was not significantly influenced by sodium. Furthermore, the shocked cells retained sodium-stimulated transport. The accumulated results suggest that the sodium-activated glutamate transport system resides in the cytoplasmic membrane and that releasable binding protein(s) is not intimately involved in its function.     

Role for Endonuclease I in the Transmission Process of Colicin E<Subscript>2</Subscript>

The bactericide colicin E₂ is believed to act by binding to surface receptors and thereby initiating the movement of periplasmic endonuclease I to the membrane or cytoplasm, with subsequent DNA degradation. Escherichia coli cells are found to become resistant to colicin E₂ by losing their endonuclease I through mutation or osmotic shock treatment.     

Two Forms of d-Glycerate Kinase in Escherichia coli

Escherichia coli K-12 synthesizes two chromatographically distinct forms of glycerate kinase which differ both in their thermolability and in the dependence of their activity upon pH. One enzymatic form, GK I, is found in cells grown with glycerate, glucarate, or glycolate. Of these compounds, glycolate is the only carbon source that elicits the synthesis of the second enzymatic form, GK II.