Genes for ribitol and D-arabitol catabolism in Escherichia coli: their loci in C strains and absence in K-12 and B strains.

Escherichia coli C strains can grow at the expense of the two natural pentitols ribitol and D-arabitol, sugar alcohols previously thought not to be utilized by E. coli. E. coli strains K-12 and B cannot utilize either compound. The genetic loci responsible for pentitol catabolism in E. coli C, designated rtl and atl, are separate and closely linked. Each lies between metG and his and is highly co-transducible with metG and with a P2 prophage attachment site. rtl and atl readily can be transduced into E. coli K-12 or B strains, in which they integrate at, or very near, their E. coli C location. Transduction also can be used to insert rtl and atl into certain E. coli K-12 F' plasmids. No recombination between E. coli C strains and either K-12 or B strains occurs within the rtl-atl genetic region after interstrain conjugations or transductions. No cryptic rtl or atl genes in K-12 or B strains can be detected by complementation, recombination, or mutagenesis. These results are consistent with the view that the rtl-atl portion of the E. coli C chromosome has no counterpart in E. coli K-12 or B and may have been obtained from an extrageneric source. Detailed biochemical and genetic comparisons of penitol utilization in E. coli and Klebsiella aerogenes are in progress. The ability to catabolize xylitol is conferred upon E. coli C strains by a mutation at or adjacent to the rtl locus, whereas in E. coli K-12 or B strains harboring rtl an additional mutation at a separate locus is required for xylitol utilization.     

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.     

Specialized peptide transport system in Escherichia coli.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

nov: a new genetic locus that affects the response of Escherichia coli K-12 to novobiocin

We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.     

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E. coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms.     

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

Integration of bacteriophage lambda into the cryptic lambdoid prophages of Escherichia coli.

Bacteriophage lambda missing its chromosomal attachment site will integrate into recA+ Escherichia coli K-12 and C at the sites of cryptic prophages. The specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. A NotI restriction site on the prophage allowed its physical mapping. This allowed us to identify the locations of Rac, Qin, and Qsr' cryptic prophages on the NotI map of E. coli K-12 and, by analogy, to identify the cryptic prophage in E. coli C as Qin. No new cryptic prophages were detected in E. coli K-12.     

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Cloning and expression in Escherichia coli K-12 of the genes for major outer membrane protein OmpA from Shigella dysenteriae, Enterobacter aerogenes, and Serratia marcescens.

The outer membranes of many gram-negative bacteria contain a major heat-modifiable protein which shows serological cross-reactivity with the OmpA protein of Escherichia coli K-12. Using the cloned gene for the E. coli K12 protein as a DNA-DNA hybridization probe, we were able to identify the corresponding genes from Shigella dysenteriae. Enterobacter aerogenes, and Serratia marcescens. These were cloned in a phage lambda vector, and their expression in E. coli K-12 was studied. All three OmpA proteins were fully produced and correctly exported to the outer membrane. In several cases, complete or partial restoration of known function of the E. coli K-12 protein was observed.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.     

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.     

A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12

The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

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.     

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.     

New Rifampin-Resistant Mutant of Escherichia coli

A rifampin-resistant ribonucleic acid (RNA) polymerase mutant, rif(r)51, derived from a presumptive RNA synthesis mutant of Escherichia coli K-12, complements rif(r) RNA polymerase mutants isolated from other strains of E. coli K-12.