Use of gene fusions to determine the orientation of gene phoA on the Escherichia coli chromosome.

We present genetic evidence which demonstrates that the phoA gene is transcribed in the clockwise direction on the Escherichia coli chromosome, in contrast to an earlier proposal. Our conclusion is based on analysis of various genetic fusions between the lac operon and the phoA gene.     

Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported.     

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Up-promoter mutations in the lpp gene of Escherichia coli.

The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the -10 and the -35 regions of the lpp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The beta-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(P1) and TATAAT (consensus sequence; P2), respectively. Similarly, it increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(R1) and TTGACA (consensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result in the highest activity. With use of the improved lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.     

Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.     

Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.     

Corrected gene assignments of Escherichia coli pro- mutations.

We have reevaluated the gene assignments of the proline mutant alleles of some known Pro- Escherichia coli strains. Of nine proline auxotrophs included in the study, five presented phenotypes inconsistent with their previously assigned genotypes. We discuss the possible sources and the consequences of these assignment errors.     

重组基因表达对大肠杆菌生理的影响

重组基因在表达外源蛋白质时常常会耗用大量的宿主细胞资源,从而对宿主造成代谢负荷,代谢负荷使得宿主的生化和生理产生很大的变化,甚至损害宿主正常的代谢功能。而过重的代谢负荷会影响目标蛋白的表达量和表达质量。综述了产生代谢负荷的原因,宿主细胞对代谢负荷的应激反应、以及减轻代谢负荷的策略。     

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.     

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.     

Effect of mutations in dnaK and dnaJ genes on cysteine operon expression in Escherichia coli

The effect of mutations indnaK anddnaJ genes on the expression of two operons that are part of cysteine regulon was determined usingEscherichia coli strains harboringcysPTWA::lacZ andcysJIH::lacZ fusions. NulldnaJ, anddnaKdnaJ mutants were impaired in β-galactosidase expression from both fusions. Effecient complementation of this defect by wild-type alleles present on a low-copy number plasmid was achieved. The presence of the pMH224 plasmid coding for CysB^* protein defective in DNA binding lowered β-galactosidase expression fromcysPTWA::lacZ fusion strain harboring wild-typednaKdnaJ alleles but did not diminish enzyme expression in ΔdnaJ and ΔdnaKdnaJ strains.