Identification of Closely Linked Loci Controlling Ultraviolet Sensitivity and Refractivity to Colicin E2 in Escherichia coli

Mutants (phenotypic symbol Ref-II) refractory to colicin E2 have been isolated in several strains of Escherichia coli K-12, and a refII locus has been mapped 1 to 2 min counter clockwise to thr. A small number of Ref-II mutants are also ultraviolet (UV)-sensitive and the uv(s) locus in one such strain has been mapped close to the refII locus near thr. The Ref-II mutation alone does not affect recombinant formation in F(-) strains, but the Ref-II, UV(s) strains behave in many respects like Rec(-) mutants, giving reduced recombination frequencies in crosses with male strains. It is suggested that the refII and uv(s) loci correspond to closely linked if not identical genes, concerned in some way in the activity of one or more deoxyribonucleases, and that the Ref-II, UV(s) mutants arise as the pleiotropic expression of a single gene or of a deletion or polar mutation affecting linked genes.     

Kinetics of Lethal Adsorption of Colicin E2 by Escherichia coli

The kinetics of lethal adsorption of colicin E2 by Escherichia coli C6 were studied by means of survivor plots. These were determined by a method which allowed rapid sampling of the reaction mixture and estimation of approximate confidence limits for the plotted data. The results were consistent with the predictions of a hypothetical model that assumed a single-hit mechanism of colicin action upon a bacterial population whose cells varied in their number of specific (lethal) receptors for colicin. The possibility of nonlethal adsorption is discussed.     

Interaction of 125I-Labeled Colicin E1 with Escherichia coli

Colicin E1 protein was labeled with ¹²⁵I to specific activities of up to 2 × 10⁸ cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.     

Isolation and Characterization of a Mutant Colicin E2

Escherichia coli K-12 colicinogenic for Col E2 yielded a mutant, SK95, that carries a nonsense mutation in the colicin structural gene. A derivative of SK95 that carries an as yet unidentified suppressor mutation produces a colicin E2 that is temperature sensitive (TS). This mutant colicin kills sensitive cells at low temperature but not at high temperature; the colicin adsorbs to cells at high temperature but does not kill them unless the temperature is lowered. Unlike normal colicin E2, which adsorbs rapidly to cells, TS colicin E2 adsorbs slowly over a period of several hours. The biochemical target of colicin E2 is deoxyribonucleic acid (DNA). When acid solubilization of DNA was compared in cells treated with either TS or normal colicin E2, striking differences were observed. Cell killing and acid solubilization of DNA by colicin E2 were shown to be separable events under certain conditions. The results are discussed in relation to the mechanism of action of colicin E2.     

YieJ (CbrC) Mediates CreBC-Dependent Colicin E2 Tolerance in Escherichia coli

Colicin E2-tolerant (known as Cet2) Escherichia coli K-12 mutants overproduce an inner membrane protein, CreD, which is believed to cause the Cet2 phenotype. Here, we show that overproduction of CreD in a Cet2 strain results from hyperactivation of the CreBC two-component regulator, but CreD overproduction is not responsible for the Cet2 phenotype. Through microarray analysis and gene knockout and overexpression studies, we show that overexpression of another CreBC-regulated gene, yieJ (also known as cbrC), causes the Cet2 phenotype.Colicins are protein antibiotics that have various modes of action. They are usually encoded on plasmids and, in many cases, alongside genes encoding colicin immunity factors, which protect colicin-producing cells from the colicin they produce. Of the enzymatic (E) colicins, some carry nuclease activity, including colicin E2, colicin E9, and colicin E3. These three proteins bind to susceptible cells via the surface protein BtuB (the vitamin B₁₂ importer) and, through a series of events that are poorly understood, cross the cell envelope to enter the cytoplasm, where they degrade nucleic acids: colicins E2 and E9 target DNA; colicin E3 targets rRNA (11).Cells can readily become tolerant of E colicins. Mutants usually have lost either the colicin receptor or some protein involved in colicin import. Loss-of-function mutations in btuB confer tolerance of high levels of colicins E2, E9, and E3. Almost 40 years ago, Escherichia coli mutants having a colicin E2-tolerant (Cet2) phenotype were identified. The Cet2 phenotype confers tolerance of colicins E2 and E9 only, while cells remain susceptible to colicin E3, and BtuB is intact (8, 9). Cet2 mutants were shown to overproduce an inner membrane protein (26), and the cet2 mutation was found to be dominant in trans and mapped at 99.9 min on the E. coli chromosome (8, 9). Using the Cet2 mutant RB208 as a source of genomic DNA, a clone able to transform E. coli cells to a Cet2 phenotype was identified. Since this clone carried a gene predicted to encode an inner membrane protein with properties identical to those overproduced in Cet2 mutants, the gene was named cet (15).The cet gene is the last gene in the four-gene cre locus, so cet is also known as creD. The other genes in this locus are creA (hypothetical open reading frame [ORF]); creB, encoding a response regulator; and creC, encoding a sensor kinase. CreB and CreC form a classical two-component regulatory system, and we recently showed that CreBC are activated upon fermentation of glucose in minimal medium or during aerobic growth on minimal medium containing fermentation products, such as pyruvate, lactate, or acetate, as the sole carbon and energy source (10). CreBC controls the expression of a number of genes (the Cre regulon), some of which encode metabolic functions but several of which are hypothetical. One of the most tightly controlled Cre regulon genes is creD (5).We have previously shown that the Cet2 strain RB208 has a point mutation in creC but that creD itself is wild type (5). Since the RB208 genomic clone capable of transforming cells to a Cet2 phenotype carries the whole cre locus, not just creD (15), our hypothesis is that the Cet2 phenotype of the transformant was due to a trans-dominant mutation in the cloned creC mutant allele activating one or more Cre regulon genes and that the Cet2 phenotype may or may not be caused by overexpression of creD. The aims of the experiments described in this paper were to test our hypothesis that the Cet2 phenotype is caused by activating mutations in CreBC and to definitively identify the Cre regulon gene that encodes the colicin E2 tolerance (Cet) protein.     

Amount of Colicin Release in Escherichia coli Is Regulated by Lysis Gene Expression of the Colicin E2 Operon

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.     

Incompatibility Exhibited by Colicin Plasmids E1, E2, and E3 in Escherichia coli

Escherichia coli strains were made multiply colicinogenic for the colicin plasmids E1, E2, or E3 (Col E1, Col E2, or Col E3, respectively) by both a deoxyribonucleic acid transformation system and bacterial conjugation. The multiply colicinogenic bacteria constructed exhibited an immunity to the colicins produced by all the plasmids they carried and also produced colicins corresponding to all the plasmids they carried. An incompatibility was observed among the plasmids. In doubly colicinogenic cells where the presence of two plasmids was established, Col E2 was lost more frequently than Col E3. In triply colicinogenic cells, Col E1, Col E2, and Col E3 were lost, with Col E3 being lost least frequently. A significant reduction in the acquisition of a conjugationally transferred Col E1 plasmid by cells colicinogenic for Col E1 was demonstrated.     

Nature of DNA and RNA Degradation in Escherichia coli Induced by Colicin E2

Lipogenesis in the fatty liver of rat, which was induced by feeding an amino acid unbalanced diet containing 8% casein supplemented with 0.3% dl-methionine, has been studied by measuring the incorporation of glycerol-1-¹⁴C, palmitate-1-¹⁴C, citrate-1,5-¹⁴C, pyruvate-1-¹⁴C and pyruvate-2-¹⁴C into various lipid fractions and ¹⁴CO₂ during in vitro incubation of liver slices.

The total radioactivity of liver lipid per 100 g of the body and the incorporation of each substrate into triglyceride in the lipid were significantly higher in the imbalance group than the control group. Conversion of each substrate to ¹⁴CO₂ was not imparied in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the triglyceride synthesis by both the fatty acid synthesis and the transesterification of fatty acid.

These results are considered to support the previous assumption in which acetate-1-¹⁴C was used as a precursor.     

基因敲除提高大肠杆菌工程菌生产异丁醇产量的研究

探究pflB、frdAB、fnr和AdhE四基因缺失突变株对大肠杆菌工程菌发酵生产异丁醇的影响。运用Red重组系统敲除大肠杆菌BW25113的pflB、frdAB、fnr和AdhE基因,构建pflB、frdAB、fnr和AdhE四基因缺失突变株E.coliBW25113H,结合本实验室已经构建的表达质粒pSTV29-alsS-ilvC-ilvD-kdcA,并检测该工程菌在1L发酵罐的发酵过程中的生物量、突变菌株的稳定性、异丁醇产量及有机酸含量的变化情况。成功获得pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H。发酵结果表明,该工程菌能以较长时间,较高比生长速率保持对数生长期,其稳定性较好,异丁醇产量增加了40%。成功构建pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H,结合非自身发酵途径使异丁醇的产量由3 g/L提升至4.2 g/L。     

大肠杆菌astE、rph基因敲除突变株的构建

目的:分别构建大肠杆菌astE、rph基因敲除突变株,并检测其异丁醇耐受性的变化。方法:利用Red重组系统分别敲除大肠杆菌的astE和rph基因,并对所获得的突变株进行异丁醇耐受性相关实验研究。结果:成功构建了astE基因缺失突变株△astE和rph基因缺失突变株Δrph,发现两种突变株的异丁醇耐受性均有所提高。结论:通过缺陷菌株的构建,为未来进一步代谢改造生产异丁醇和研究异丁醇耐受机制奠定了基础。     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.     

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

A Mutant Escherichia coli Primase Defective in Elongation of Primer RNA Chains

Earlier we showed by affinity cross-linking of initiating substrates to Escherichia coli primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A. A. Mustaev and G. N. Godson, J. Biol. Chem. 270:15711-15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5' CTG 3' pRNA initiation site on phage G4 oric DNA. The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.     

Mutant Strains of Escherichia coli K-12 Exhibiting Enhanced Sensitivity to 5-Methyltryptophan

Eighteen mutants (designated MT(s)), isolated in Escherichia coli K-12, showed increased sensitivity to inhibition of growth by 5-methyltryptophan. All mutants were also much more sensitive to 4-methyltryptophan and 7-azatryptophan but exhibited near normal sensitivity to 5-fluorotryptophan and 6-fluorotryptophan. All of the mutations were linked to the trp operon. Their locations within the trp operon were established by deletion mapping. There was good agreement between the map position of an MT(s) mutation and a lowered activity of one of the tryptophan pathway enzymes. Three mutants, one of which contained a mutation that mapped within the trpE gene, were deficient in their ability to use glutamine as an amino donor in the formation of anthranilic acid. Another trpE mutation led to the production of an anthranilate synthetase with an increased sensitivity to feedback inhibition by tryptophan.     

Colicin pore-forming domains bind to Escherichia coli trimeric porins

Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.     

Reversibility of the Specific Adsorption of Colicin E2-P9 to Cells of Colicin-Sensitive Strains of Escherichia coli

The adsorption of colicin E2-P9 to its specific receptors on cells of sensitive strains of Escherichia coli is reversible under normal experimental conditions. At temperatures above 20 C, colicin may desorb from one cell and be readsorbed by a second with potentially lethal consequences. However, desorption of colicin seems unable to rescue a cell once it has received a lethal dose. These findings have implications both for the nature and types of specific receptors, and for the assay of colicin by the survivor count (lethal unit) methods.