Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.     

Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage

Phage display is a widely used method to optimize the binding characteristics of protein-ligand interactions. In addition, it has been used to clone genes from genomic and cDNA libraries based on their ligand-binding characteristics. One difficulty often encountered when expressing heterologous proteins by phage display is the toxicity of the protein on the Escherichia coli host. Previous studies have shown that heterologous protein expression can be tightly controlled using plasmids with the P(BAD) promoter of the arabinose operon of E. coli, and the araC gene, which is both a positive and negative regulator of the promoter. We constructed a set of phage display vectors that utilize the P(BAD) promoter to control the expression of proteins on the surface of the M13 bacteriophage. These vectors exhibit tightly controlled expression of proteins on the surface of the phage. In addition, the amount of protein displayed on the phage is modulated by the amount of arabinose present in the growth medium during phage propagation. This may be useful for altering the stringency of binding enrichment during phage display.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.     

Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture

The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.     

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.     

Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli

Aim: To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P_BAD promoter in Escherichia coli. Method and Results: The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose P_BAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring P_BAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions: The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study: We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes.     

Mutants with base changes at the 3'-end of the 16S RNA from Escherichia coli. Construction, expression and functional analysis

The functionally important 3' domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid-encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U. Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the lambda PL promoter. In a strain coding for the temperature-sensitive lambda repressor cI857 the mutant RNAs could be expressed conditionally. We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees. The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3' domain of the 16S RNA for ribosomal function.     

A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis.

We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.     

Site-specific mutagenesis of Escherichia coli gltT yields a weak, glutamic acid-inserting ochre suppressor

Of all the Escherichia coli tRNA genes that can give rise to an amber or an ochre suppressor by a single-nucleotide mutation, only the tRNAGlu genes have not been observed to do so. A study of the relationship between the sequences of tRNAs and the codons they translate predicts that the ochre suppressor derived from tRNAGlu would function very poorly on the ribosome. We have used site-specific mutagenesis to create the gene for such a tRNA in order to test this prediction. We cloned the tRNAGlu-Suoc gene into a high copy number plasmid, under control of the lacUV5 promoter. The mutant tRNA suppresses both amber and ochre nonsense mutations. As predicted, it is less efficient than other suppressors expressed under similar conditions.     

The arabinose kinase,ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family

The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene, ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.     

A tightly regulated inducible expression system utilizing the fim inversion recombination switch

The fim inversion system of Escherichia coli (E. coli) can behave as a unidirectional switch in an efficient manner. We have developed a new expression system for E. coli, comprising the arabinose-inducible fimE gene and the fim invertible DNA segment containing a constitutively active promoter. In this system, the target gene is cloned with the promoter in the OFF orientation, resulting in no transcribed product. When induced by arabinose, the active promoter is switched to the ON orientation via FimE-catalyzed DNA inversion, and the gene is expressed. Our expression system exhibited very tightly controlled basal expression and high induced expression, with simple induction by inexpensive arabinose. These characteristics make our system suitable for large-scale expression or for production of toxic proteins.     

Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression.

Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4. We cloned the lit gene from wild-type E. coli and three independent lit mutants. We present evidence that lit mutations [renamed lit(Con) mutations] cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression. We also present evidence that the lit gene product is nonessential for E. coli growth, although the gene is common to most E. coli K-12 strains.