Bacterial expression system with tightly regulated gene expression and plasmid copy number

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.     

A system for insertion of heterologous DNA into chromosome of group H streptococci

A system for insertion of genes into the chromosome of group H streptococci has been elaborated. It consists of a recipient strain (Gallis GS10/1), having the fragments of lambda L-47-1 bacteriophage DNA inserted into the chromosome, and lambda 202 vector. The constructed system suggests the preliminary cloning of genes in E. coli cells with their subsequent insertion into the chromosome of group H streptococci.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

A phasmid shuttle vector for the cloning of complex operons in Salmonella

Phasmid (phage plasmid hybrid) P4 vir1 can be propagated in Escherichia coli as a helper-dependent lytic phage, as a plasmid, or as a prophage. On the basis of an understanding of these modes of propagation, derivatives of P4 have been constructed for use as cloning vectors. In this report we demonstrate that phasmid P4 (i) will propagate as a helper-dependent lytic phage and as a plasmid in Salmonella spp. and (ii) can be used as a high efficiency phage shuttle vector for the reversible transfer of cloned genes between Salmonella spp. and E. coli. For both E. coli and Salmonella spp., P4 phage-mediated gene transfer proved to be only 10-fold lower than plaquing efficiency. For the case of Salmonella spp., this frequency is ca. 10(4)-fold more efficient than is typically found for the transformation of DNA molecules. The usefulness of this cloning vector system for analyses of pathogenic virulence factors is demonstrated by the cloning and expression of both the P pilus adhesin operon and the hemolysin operon of uropathogenic E. coli.     

A genetic study of Escherichia coli strains carrying Mu-lambda-Mu structures

We have studied the interaction of bacteriophages Mu and lambda after their simultaneous induction and the influence of lambda on Mu-dependent mobilization of the E. coli chromosome by the RP4 plasmid. Heterolysogenic E. coli strains carrying Mu-lambda-Mu structures were constructed (Faelen et al. 1975). The Mu and lambda prophages are linked in such structures, and the functions of some lambda genes are disturbed depending on the integration site. A study of the inhibition of Mu growth by lambda after their simultaneous induction was performed and the region of the lambda genome (R-H) which contains the gene(s) responsible for the inhibitory effect of lambda on Mu was identified. The efficiency of Mu-dependent mobilization of the bacterial chromosome by RP4 is shown to be an order of magnitude lower in strains with unlinked Mu and lambda and an order of magnitude higher in strains with some permutations of the lambda prophage than in the control Mu-monolysogenic E. coli strain. Thus the effect of Mu on mobilization depends on the localization of the lambda prophage and on the functioning of its genome within a Mu-lambda-Mu structure. It is presumed that the mobilization of the bacterial chromosome is stimulated by effective replication of the Mu genome starting from the ori site (origin of replication) of the lambda prophage within the Mu-lambda-Mu structure. We propose a model to explain the interaction of Mu and lambda in E. coli strains carrying Mu-lambda-Mu structures.     

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.     

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.     

Stimulation of mutations suppressing the loss of replication control by small alcohols

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.     

An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1.

A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.     

High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region

An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed. We have designed a PvuII site immediately behind the untranslated region. A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression. This vector also contains 7 additional cloning sites downstream from the PvuII site. A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression. We have also cloned the lambda cl gene into a p15A plasmid. Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E. coli strain. Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E. coli DH5 alpha.     

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.     

Development of broad-host-range vectors for expression of cloned genes in Pseudomonas

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.     

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.     

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.     

Structure of cryptic lambda prophages

When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.