Genetic Mapping in Escherichia coli K-12 by Radiation-Induced Crossing-Over

Conventional methods for chromosomal mapping in Escherichia coli are (i) interruption of matings to obtain minimum marker entry times, (ii) linkage analysis of recombinants, and (iii) cotransduction. Method (i) has a resolution of about 0.5 min (5 x 10(4) nucleotides) and is not useful for distances less than about 1 min; methods (ii) and (iii) are capable of better resolution but are generally not very reproducible and no general theory is available for translating crossing-over and cotransduction frequencies into physical chromosomal distances. We found that when merozygotes are irradiated (X rays or ultraviolet light) soon after marker transfer, high linkage values (0.8 to 1.0) between nearby marker pairs decrease with radiation dose to 0.5. Our results are quantitatively consistent with the idea that radiations induce crossing-over lesions proportional to dose, and the number of such lesions between two markers is proportional to the physical separation of the markers in the range that can also be measured by interruption of mating (0.5 to 4.0 min). Additivity relations among markers are also satisfied. We used this technique to measure the distances (0.1 to 1.0 min) between several pairs of closely linked markers.     

Mapping of nrdA and nrdB in Escherichia coli K-12.

The structural genes coding for the B1 and B2 subunits of the enzyme ribonucleoside diphosphate reductase, nrdA (formerly designated dnaF) and nrdB, respectively, have been mapped in Escherichia coli. They are located at approximately 48 min. The gene order in this region of the E. coli chromosome was found to be purF glpT nrdB nrdA nalA cdd dcd his.     

Genetic Mapping of the minB Locus in Escherichia coli K-12

The minB (minicell production) locus of Escherichia coli K-12 was mapped by transduction using bacteriophage P1. minB is located at min 25.6, between purB (min 25.2) and dadR (min 25.8). The mapping was facilitated by the use of insertion zcf-236::Tn10, which is inserted at min 25.4.     

Keratinocyte growth factor-2 production in an ompT-deficient Escherichia coli K-12 mutant

A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein. This cleavage resulted in a truncated KGF-2 species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity. This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl. To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation. Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser). From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavage issue.     

More Precise Mapping of the Replication Origin in Escherichia coli K-12

The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.     

Rapid Mapping of Conditional and Auxotrophic Mutations in Escherichia coli K-12

The approximate genetic map locations of auxotrophic and conditional lethal mutations of Escherichia coli can be rapidly determined with replica plating techniques. A set of patches of 15 streptomycin-sensitive (Str^S) Hfr strains with points of origin distributed around the map is replica plated onto a recombinant-selective plate with a lawn of Str^R cells which carry an unmapped mutation. The map interval defined by the Hfr points of origin which are closest to the mutant locus is seen by the presence or absence of heavy patches of recombinants produced by transfer of early wild-type genes from the Hfrs. An alternative method is to replicate patches of different mutant strains (100 per plate) onto Hfr lawns; in this case more than 1,000 different mutants can be mapped in a single experiment in a few days. In this way, many types of mutations with similar phenotypes can be grouped as to approximate location on the genetic map. For ordering mutations within groups, the same replica plating methods can be used to cross F-prime derivatives of mutants with other mutants of the same group. Relative merits of these and other mapping methods of E. coli are discussed.     

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     

Influence of mutations at genes of global transcriptional regulators on production of autoinducer AI-2 Quorum Sensing in the system of Escherichia coli

The control of gene expression in response to an increase in the bacterial population density (Quorum Sensing) involves low-molecular-weight signal molecules (autoinducers, AI). AI-2 and synthase LuxS mediating its synthesis are widely distributed in Gram-negative and Gram-positive bacteria. In this work, the data were obtained on the role of global regulators of gene expression in AI-2 synthesis in Escherichia coli cells. The mutation inactivating gene rpoS (encodes sigma S subunit of RNA polymerase) was shown to drastically decrease an amount of active AI-2 in the culture medium. Mutations at gene rpoN that encodes sigma N subunit of RNA polymerase and also at gene lon, which encodes Lon proteinase, on the contrary, increase an amount of active AI-2 in supernatants of cultures. Mutant strains lacking histone-like proteins H-NS and StpA accumulate a slightly higher amount of AI-2 than the isogenic wild-type strain: however, an amount of AI-2 decreased in the culture medium of the double mutant devoid of both these proteins.     

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.     

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

Phosphoglucomutase Mutants of Escherichia coli K-12

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon.     

Methyl-cytosine specific restriction in Escherichia coli K-12

Experiments on transformation of Escherichia coli K-12 cells by plasmids carrying RM systems with different recognition sites containing 5-methylcytosine have shown that the gene mcrB determines the function of restriction. The data obtained made it possible to believe that E. coli possesses no restriction system recognizing specifically cytosine methylated in position 4.