Characterization of an Escherichia coli lysA insertion targeted mutant using phenotype arrays

The objective of this study was to investigate the effect of a lysine biosynthesis insertion mutation on the growth response and phenotype of Escherichia coli. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. This E. coli insertion mutant exhibited altered growth physiology and phenotype of the recipient E. coli. The constructed mutant could grow in the absence of lysine supplementation although the extent of growth after 7 h incubation in the presence of most lysine concentration was significantly (p<0.05) decreased compared to that observed with the parent E. coli strain. The mutant was also less able to utilize carbon and nitrogen substrates than the parent E. coli strain as determined by using phenotype arrays. These results suggest that the carbon and nitrogen phenotype profiles of E. coli when measured on phenotype arrays are altered after targeted insertion mutagenesis in the lysA gene. Creation of altered phenotypes may have potential for pharmaceutical and biotechnological applications of lysine E. coli metabolism.     

The product of gene secC is involved in the synthesis of exported proteins in E. coli

To obtain additional mutants in the secretory apparatus of E. coli we have isolated suppressors of a mutant (secAts) that is temperature-sensitive for secretion. One of these, secC, can suppress the secretion defect of secA and has a phenotype of its own. At 23 degrees C, the secC mutant is cold-sensitive for growth and blocks the synthesis of transported proteins. The synthesis of at least one secreted protein, maltose-binding protein (MBP), can be restored by mutations that alter the hydrophobic region of the signal sequence of MBP. The phenotype of the secC mutant suggests that the SecC protein may be a component of the secretory apparatus of E. coli; it also supports the notion that in procaryotes secretion and gene expression are coupled. The secC gene maps at 68.5 minutes on the E. coli chromosome.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless cI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsB(ts) mutant. The analysis of the ribosomes isolated under the non-permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non-permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the "DeaD/CsdA" DEAD-box helicase, has been isolated as a multicopy suppressor of rpsB(ts) mutations. Here, we show that expression of a plasmid-borne DeaD/CsdA gene restores both S1 and S2 on the ribosome at the non-permissive temperature in the rpsB(ts) strain, which in turn leads to suppression of the translational defect affecting canonical mRNSa. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.     

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.     

The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.

Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures. The cloned gene was sequenced, mapped to the 20.9-min region of the E. coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.     

Translational coupling between the ilvD and ilvA genes of Escherichia coli.

The hypothesis that translation of the ilvD and ilvA genes of Escherichia coli may be linked has been examined in strains in which lacZ-ilvD protein fusions are translated in all three reading frames with respect to ilvD. In these strains, the nucleotide sequence was altered to obtain premature termination of ilvD translation, and in one strain translation termination of ilvD DNA occurred two bases downstream of the ilvA initiation codon. In the wild-type strain, the ilvD translation termination site was located two bases upstream of the ilvA start codon. In each of the mutant strains, expression of ilvA, as determined by the level of threonine deaminase activity, was strikingly lower than in the wild-type strain. The data suggest that expression of ilvD and ilvA is translationally coupled. By inserting a promoterless cat gene downstream of ilvA, it was shown that the differences in enzyme activity were not the result of differences in the amount of ilvA mRNA produced.     

λN gene expression regulated by translation termination in ribosome L24 mutant

Although the initiation and elongation steps of protein synthesis have been extensively char-acterized in Escherichia coli (E. coli), the translation termination is still less well understood. However, recent experiment result might have provided some hints for our deeper understanding of the termination mechanism. (i) Not only does the translation stop codon act as a signal for ter-mination, but also its context influences the translation termination[13]; (ii) the structure similar-ity betwee…     

枯草芽胞杆菌蛋白质表达分泌系统发展及展望

枯草芽胞杆菌作为革兰阳性模式菌株是基础研究和工业应用的常用宿主细胞。介绍了枯草芽胞杆菌中蛋白合成和分泌过程中的重要步骤及重要调控位点。在枯草芽胞杆菌蛋白表达及分泌系统中,可以针对目标基因在体内的转录、翻译、折叠、转运和菌株改造等方面对表达分泌系统进行优化改良,针对不同的目标蛋白,可进行不同优化模块的组装和拼搭,以达到针对目标蛋白产物定制化地提高产量和分泌量的目的。在未来,随着基因编辑和合成生物技术的发展,菌株改良策略的不断优化,枯草芽胞杆菌将会在工业生产蛋白质制品领域发挥更大的应用价值。     

Silent mutations result in HlyA hypersecretion by reducing intracellular HlyA protein aggregates

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. Extracellular protein secretion has the advantage of reducing protein aggregation and simplifying downstream purification. The introduction of five rare codons in a specific region of the alpha-hemolysin (hlyA) gene previously was shown to result in eightfold improvement in secretion of HlyA via the hemolysin (Type-I) pathway. Here we investigate the biological basis for the observed phenomenon that translation rate of HlyA protein may be related to the ability to secrete higher levels of HlyA via the Type-I pathway. A detailed comparative analysis between a hypersecreter mutant strain (hly-slow) and a control strain (hly-parent) shows a significant decrease (by approximately 50%) in the intracellular level of HlyA protein in the hly-slow strain relative to the hly-parent strain. Nearly 100% of the intracellular HlyA protein exists in the inclusion body fraction in both the strains. These results demonstrate the importance of synonymous codon changes in the context of improving HlyA secretion yield via Type-I pathway and further illustrate that production of high levels of secreted proteins appears to require a balance between translation and secretion rate.     

Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.     

Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein.

A Pseudomonas aeruginosa outer membrane protein F-deficient omega-insertion mutant strain H636, in contrast to its protein F-sufficient parent strain H103, was unable to grow on unsupplemented Proteose Peptone no. 2 broth (Difco Laboratories, Detroit, Mich.). Addition of high concentrations of NaCl, KCl, glucose, sucrose, or potassium succinate permitted growth of strain H636 at rates approaching those of the parent strain H103. Strain H636 cells were 33% shorter and had a 46% smaller cross-sectional area than did the parent strain growing at similar rates on the same medium. These properties of the oprF::omega mutant were analogous to those previously observed for Escherichia coli ompA mutants in an lpp (Braun lipoprotein-deficient) mutant background. Therefore, we compared P. aeruginosa protein F and the E. coli OmpA protein. In addition to many similarities previously described, sequence alignment demonstrated substantial amino acid sequence homology throughout the carboxy-terminal 168 to 180 amino acids of the two proteins. Consistent with this observation, polyclonal antiserum specific for OmpA reacted on Western blots (immunoblots) with protein F. Expression of protein F from the cloned oprF gene in an E. coli ompA lpp double mutant resulted in a 1.7-fold increase in cell length and a 2.1-fold increase in cross-sectional area compared with values for the same mutant containing only the plasmid vector onto which the oprF gene had been cloned. These results favor a structural role for P. aeruginosa protein F and suggest that it is strongly related to the E. coli OmpA protein.