Mutations in the Escherichia coli RNA-polymerase beta-subunit gene cloned in a multicopy plasmid

A multicopy plasmid pLMN1 expressing a wild type rpoB gene encoding Escherichia coli RNA polymerase beta subunit gene was constructed. Introduction of this plasmid into rifampicin-resistant RpoB mutants makes them rifampicin-sensitive. Rifampicin-resistant clones appear in such strains with frequencies up to 10(-3), due to recombinational (recA-dependent) transfer of rif-r mutations from chromosome to pLMN1. This provides a simple selection procedure for transfer of any rpoB mutation, together with a rif-r mutation from a chromosome to pLMN1. In this way, we transferred rpoB22 amber mutation to pLMN1 for localization of the mutant codon by DNA sequencing.     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.     

Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway

Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247-261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985;27:1668-1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328-338), chromosomal insertion of the gene which product is desired, or changing the plasmid-coded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624-628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952-960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h(-1) (uninduced) to 0.64 h(-1) (induced). The same transformation in an Escherichia coli zwf(-), allows a growth-rate recovery from 0.43 h(-1) (uninduced) to 0.62 h(-1) (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h(-1) in the TrpLE-proinsulin induced strain, to 0.37 h(-1) when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression.     

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

The use of the multicopy plasmid pUC19 for assuring the constitutive expression of gene rplL in Escherichia coli

A mutation in lac-operator region of pUC19 plasmid causing an increase in beta-galactosidase activity was observed. The plasmid was used as a vector to provide high level of expression of the cloned E. coli rplL gene.     

Analysis of the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in Escherichia coli

The antimutagenic effect of cinnamaldehyde on mutagenesis was investigated using ten kinds of chemical mutagen in Escherichia coli WP2s (uvr A-). In addition, the frequency of mutation induction by each mutagen in an SOS repair deficient (umuC-) strain was compared with that in a wild-type (umuC+) strain. Cinnamaldehyde greatly suppressed the umuC-dependent mutagenesis induced by 4-nitroquinoline 1-oxide (4-NQO), furylfuramide or captan. However, cinnamaldehyde was less effective against the umuC-independent mutagenesis by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate. On the other hand, no inhibitory effect of cinnamaldehyde was observed on prophage induction or tif-mediated filamentous growth. These results suggest that a cinnamaldehyde does not prevent the induction of the SOS functions. Despite the decrease in the number of revertants, a remarkable increase was observed in the survival of 4-NQO-treated WP2s cells after exposure to cinnamaldehyde. The reactivation of survival suggests the promotion of some DNA repair system by cinnamaldehyde. This enhancement of survival was also observed in uvr B, polA, recF or umuC mutants and less in lexA or recB, C mutants. However, it was not observed in recA mutants. Therefore, we assume that cinnamaldehyde may enhance an error-free recombinational repair system by acting on recA-enzyme activity.     

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4

Summary A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30° C but not at 42° C, clones are isolated resistant to kanamycin at 42° C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30° C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42° C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained.About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10^-10.The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn₁ in many Gram-negative bacterial species.     

Curcumin inhibits the SOS response induced by levofloxacin in Escherichia coli

The role of RecA protein in bacterial resistance to antibiotics makes this protein attractive from a pharmacological point of view. In this study we demonstrate that curcumin is able to inhibit the SOS response in Escherichia coli induced by levofloxacin. The bla_TEM-1 gene has been placed under the control of the LexA-binding box and used as reporter gene. The expression of TEM-1 β-lactamase enzyme was increased in the presence of ssDNA induced by levofloxacin, while, the presence of curcumin at 8 μg/ml, reduced dramatically the expression of the reporter gene. Moreover a simple microplate assay suitable for high-throughput screening has been developed.     

Mathematical model of induced mutagenesis in bacteria Escherichia coli under ultraviolet irradiation

A mathematical model is developed for a mutational process in bacteria Escherichia coli induced by ultraviolet radiation. The dynamics of the basic protein complexes of the SOS-response system is investigated. The probability of mutations during translesion synthesis is estimated.     

Involvement of Escherichia coli K-12 recombination functions in elimination of multicopy plasmid R6K by DNA-damaging agents

Abstract A neutral lipomannan has been isolated from the membranes of Micrococcus agilis . In contrast to the lipomannans from 2 strains of Micrococcus luteus , which contained succinic acid ranging from 5.1%–8.0%, the M. agilis lipomannan had no detectable succinyl residues and exhibited neutral behaviour on Concanavalin A-agarose rocket electrophoresis. As with the M. luteus lipomannans, mannose was the only sugar detectable (as alditol acetate) by GLC analysis in the purified M. agilis lipomannan. Fatty acids accounted for 2% of the M. agilis lipomannan and were predominantly C₁₅ branched-chain acids, with higher amounts of C₁₆ iso and C₁₇ anteiso than that found in the M. luteus polymers. Neither conditions of growth of the organism nor the method of membrane preparation appeared to be responsible for the absence of succinyl residues. This appears to be the first report of a neutral membrane amphiphile.     

Sodium arsenite inhibits spontaneous and induced mutations in Escherichia coli

Sodium arsenite at a non-toxic concentration was found to inhibit strongly mutagenesis induced by ultraviolet light (UV), 4-nitroquinoline-1-oxide (4NQO), furylfuramide (AF-2) and methyl methane-sulfonate (MMS) as well as spontaneous mutation in the reversion assay of E. coli WP2uvrA/pKM101. The effect was not, however, seen in the case of the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In order to elucidate the mechanism of the mutation-inhibitory effect of sodium arsenite, its action on umuC gene expression and DNA-repair systems was investigated. It was found that sodium arsenite depressed beta-galactosidase induction, corresponding to the umuC gene expression. For UV-irradiated E. coli strains possessing different DNA-repair capacities, sodium arsenite decreased the UV survival rates of WP2, WP2uvrA[uvrA] and WP67[uvrA polA], increased those of SOS-uninducible strains having either the recA+ or uvrA+ such as CM571 [recA], CM561 [lexA(Ind-)] and CM611[uvrA lexA (Ind-)], and did not affect that of the uvrA recA double mutant, WP100. From these results, we assume that sodium arsenite may have at least two roles in its antimutagenesis: as an inhibitor of umuC gene expression, and as an enhancer of the error-free repairs depending on the uvrA and recA genes.     

Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli

We used preS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43 degrees C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-beta-galactosidase aggregation at 43 degrees C. Overexpression of PIMT reduces the amount of preS2-S'-beta-galactosidase found in inclusion bodies at 43 degrees C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-beta-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.     

Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

A rapid site-directed mutagenesis strategy using homologous recombination and DpnI digestion of the template in Escherichia coli is described. Briefly, inverse polymerase chain reaction amplification of the entire circular plasmid was performed by mutagenic primers with overlapping sequences ( approximately 15 bp) for generating PCR products with approximately 15 bp of homology on the terminal ends. On direct transformation of the amplified PCR products into restriction endonuclease DpnI-expressing E. coli BUNDpnI, homologous recombination occurs in E. coli while the original templates are removed via DpnI digestion in vivo, thus yielding clones harboring mutated circular plasmids. Nearly 100% efficiency was attained when this strategy was used to modify DNA sequences.