Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation

Escherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.     

Effects of paraquat on Escherichia coli: differences between B and K-12 strains.

Escherichia coli B and K-12 are equally susceptible to the bacteriostatic effects of aerobic paraquat, but they differed strikingly when the lethality of paraquat was evaluated. E. coli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E. coli K-12 did not. This difference depended on the ability of the B strain, but not the K-12 strain, to retain internalized paraquat; the B strain was killed on aerobic tryptic soy-yeast extract plates during the incubation which preceded the counting of colonies. This difference in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by cyanide-resistant respiration after brief exposure to paraquat, washing, and testing in fresh medium. This difference was also shown by using [14C]paraquat. This previously unrecognized difference between E. coli B and K-12 has been the cause of apparently contradictory reports and should lead to some reevaluation of the pertinent literature.     

Rapid and accurate identification of Escherichia coli K-12 strains.

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.     

Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake.

Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an 11-kilobase E. coli DNA fragment containing fepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products. The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparently had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown. The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.     

Isolation and characterization of mutator strains of Escherichia coli K-12.

A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation. Three types of mutants were isolated. Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3. The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR. mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations. The mutator phenotype is recessive. Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans. No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain. The mutator's activity was little affected by the type of medium in which the strain was grown. Deoxyribonucleoside triphosphate pools were normal in mutR34. Intergenic recombination frequencies were the same in mutR and mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr times Fminus crosses when the recipeint was mutR34 as compared with mutR+. This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.     

Polymorphism in the dgt-dapD-tsf region of Escherichia coli K-12 strains.

Restriction analysis of the dapD region cloned from several strains of Escherichia coli, revealed a restriction-fragment length polymorphism (RFLP). This RFLP, which includes the BamHI, EcoRI and SalI sites, may be useful in classification of various E. coli strains.     

Origin of Escherichia coli K-12 Hfr B7.

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Purification and characterization of aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Osmoregulatory expression of porin genes in Escherichia coli: a comparative study on strains B and K-12

Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium. In contrast, E. coli B produces only the OmpF porin, regardless of the medium osmolarity. In this study, it was revealed that there is an extensive deletion within the ompC locus of the E. coli B chromosome. Cloning and nucleotide sequencing of the regulatory gene, ompR , of E. coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E. coli K-12. It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E. coli B.     

Orotic acid excretion in some wild-type strains of Escherichia coli K-12.

During rapid growth, the excretion of pyrimidines, predominantly uracil, is a common phenomenon in procaryotes and eucaryotes. In Escherichia coli, some K-12 strains excrete orotic acid and not uracil. This is caused by a mutation in the pyrF gene.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

Summary In this article we describe some in vivo properties of a coldsensitive ribosomal mutant from Escherichia coli. The mutation affects the rplV gene which is the structural gene of ribosomal protein L22.Our work shows that at 22°C, the biosynthesis of both ribosomal subunits and the maturation processing of 16S and 23S ribosomal RNA are impaired. Integration of our results in a general model of in vivo ribosomal assembly in E. coli is presented.