Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid.

To determine whether the fip gene of Escherichia coli, which is required for filamentous phage assembly, is required for cell viability, we replaced the chromosomal copy of the gene with an inactive copy introduced on a plasmid. We found that the fip gene is dispensable. The method we devised, which should be generally useful, was also tested with an inactivated rho gene. As expected, the rho gene is essential.     

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.     

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.     

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.     

Immune electron microscope localization of RNA-polymerase on the Escherichia coli chromosome using monoclonal antibodies against the beta-subunit

Possibility of the immunoelectron microscopic visualization of RNA polymerase on the Escherichia coli chromosome with monoclonal antibodies against the beta-subunit labelled by [protein A.gold] complex was demonstrated. Using this method RNA polymerase molecules were revealed within nucleoid as well as on the membrane-free chromosome.     

Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification

Summary Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1: pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for -glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded -glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded -glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of -glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus -glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.     

Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli

Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.     

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.     

Role of the beta-subunits of Escherichia coli RNA-polymerase in the regulation of gene activity

The DNA-dependent RNA-polymerase from E. coli B/r and its rif-r mutant rpoB409 with pleiotropic effect has been studied. It was shown, that multiple forms of promotor sites in T4- and T7-DNA "early" regions are recognized with different efficiences by RNA-polymerases from E. coli B/r and rpoB409. The rif-r rpoB409 mutation has been reported to affect the beta-subunit. Thus, the present data indicates that the selection of promoter sites can be controlled by the beta-subunit of RNA-polymerase.     

One-step gene amplification by Mu-mediated transposition of E. coli genes to a multicopy plasmid

A general in vivo method to amplify the number of copies of a specific gene in one step is described. The method is directly applicable to any selectable gene of Escherichia coli and is based on the Mu-mediated transposition of segments of host chromosomes into the conjugative, multicopy plasmid R6K. Using this method we have cloned the β-hydroxydecanoyl thioester dehydrase structural gene, fabA, into the R6K plasmid. Strains carrying the resultant plasmid produced 13 to 21 times more dehydrase than control strains.     

Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12

The four general transaminases of Escherichia coli K-12 have overlapping, but discrete, substrate specificities and participate in the final step in the synthesis of at least seven different amino acids. Through the use of strains that have mutations in one or more transaminase genes and carry a different wild-type (wt) gene on a multicopy plasmid, it was possible to detect instances in which an amplified wt gene suppressed nonallelic mutations. In these cases, overproduction of the enzyme permitted a broader range of substrates to be used at physiologically significant levels, either because a low catalytic efficiency (in the case analyzed here) or a low affinity of the enzyme towards the substrate prevented its effective utilization under normal conditions. Consequently, by compensating for a low catalytic reaction rate, enzyme overproduction circumvents the original lesion and restores biosynthetic activity to the mutant strain. The suppression of a mutation in one gene by amplified copies of a different wt gene is termed 'multicopy suppression'. This phenomenon is useful for detecting poorly expressed genes, for detecting duplicate genes, for identifying secondary functions of the products of known genes, and for elucidating the metabolic role of the product of the suppressed gene.     

Regulation of expression of the cloned ada gene in Escherichia coli

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids. O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents. When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced. Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000. We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression. beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents. The induction was autogenously regulated by the ada gene product, in a positive manner.     

Expression of cloned calf prochymosin gene sequence in Escherichia coli

An expression plasmid for calf prochymosin (prorennin) cDNA was constructed. The plasmid (pCR301) contains the lacUV5 promoter in front of the fused gene in which the codons for the N-terminal four amino acids of prochymosin cDNA were replaced with those for the N-terminal ten amino acids of beta-galactosidase. Synthesis of the fused protein with the expected Mr was detected immunologically in Escherichia coli harboring pCR301. The product seemed to be localized in the cell membrane of the bacterial host.     

Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polylinker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Bal31. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme IIScr) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scrB, scrA, scrR. An amplified sucrose-inducible gene product, Mr 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrB, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis, Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae.