Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Stable multicopy integration of vector sequences inHansenula polymorpha

Plasmids without an origin of replication, but bearing theURA3 gene ofSaccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously inHansenula polymorpha, indicating that parts of theS. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function inH. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued inE. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into theH. polymorpha genome. The integration event usually occurred in high copy number (approx. 30–50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. It contrast, the use of homologousURA3 gene under similar conditions led to low-copy-number transformants.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii,maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

An unusual suicidal interaction inEscherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β-subunit of RNA polymerase was obtained inEscherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying otherpolA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3’, 5’-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns ⁺-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide.     

Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Expression of an endoglucanase gene fromClostridium cellulolyticum inEscherichia coli

Summary A gene coding for an endoglucanase from the anaerobic cellulolytic bacteriumClostridium cellulolyticum has been cloned by direct selection inEscherichia coli, using the carboxymethyl cellulose-Congo Red assay. The cloned gene has been subcloned in the two possible orientations in pUC plasmids. One of the two resulting constructs exhibited a higher level of expression, which was associated with a high level of plasmid instability. The enzyme synthesized inE. coli from the cloned gene has been characterized by two procedures, maxicells and gel filtration chromatography, as a polypeptide of approximately 40 kilodaltons.     

Identification of genes specifying proline biosynthesis in marine bacteriumAlteromonas haloplanktis

Summary A gene bank of total DNA of a marine bacterium,Alteromonas haloplanktis, was constructed on pBR322. Two hybrid plasmids pUS2010 and pUS2011 carrying inserts of 8.2 and 5.7 kb, respectively, were isolated that complemented theproBA deletion inE.coli CSH26. Restriction map of the inserts showed that both plasmids in common carried a 5.7 kb fragment. This restriction fragment thus contains both the genes involved in proline biosynthesis inA.haloplanktis and could be expressed inE.coli.     

Complementation of anAspergillus nidulans mutation by a gene from the basidiomyceteCoprinus cinereus

TheCoprinus cinereus acu-7 gene, encoding isocitrate lyase, has been cotransformed into strains ofAspergillus nidulans carrying anacuD mutation in the corresponding structural gene. Transformants with high copy numbers of integratedacu-7 sequences were able to grow on acetate as the sole carbon source, indicating complementation of theacuD mutation. Northern blot analysis showed that theC. cinereus gene was transcribed constitutively and not regulated normally by acetate.     

Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMB 9871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111 units g^–1 cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-(γ)-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.