Functions antagonistic to plasmid formation by lambda N- chromosomes

It was concluded in the preceding paper that lambda N- cI- genomes probably failed to form plasmids within infected Escherichia coli cells because they leakily express functions that act to destabilize the plasmid state. This prediction was investigated by examining the effect upon plasmid-forming ability of the loss of possible anti-plasmid functions. The loss of Ter function was found to allow long-term plasmid formation, although the efficiency of initial plasmid formation and the heritable stability without selection were low. The combined loss of the int, red and gam gene functions also promoted plasmid growth, although the absence of Ter lambda was necessary. In contrast, the presence of Ter80 (due to an h80 substitution) did not prevent plasmid formation when the int, red and gam genes were absent, indicating that Ter80 does not attack the closed-circular form of the lambda chromosome. The combined loss of the ter, int, red and gam gene functions facilitated fully efficient inheritance of the lambda N- cI- plasmids in the absence of selection, although the efficiency of initial plasmid formation remained low. However, cells harbouring such plasmids suffered a decline in viability, indicating that the plasmids expressed a function (or more than one) that acts to debilitate the host cells--presumably an effect that is increased with this genotype because the modified lambda N- cI- plasmids are inherently more stable. The possible involvement of the lambda S and kil functions in destabilization is discussed.     

Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes

Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.     

Vectors--derivatives of phage lambda for construction and analysis of genome libraries

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.     

Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration.

The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.     

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.     

λ Mutants Which Persist as Plasmids

Lambda phages mutated in gene N do not kill sensitive host bacteria, but persist as plasmids. Plasmids are formed by genomes containing cI(+), and also by sus, ts, or c mutants of cI. Bacteria infected with two or more phage particles give rise to clones in which most of the bacteria are carriers. The introduced lambda genomes replicate more than once per bacterial division until there are 10 to 20 lambda plasmids per host genome. In bacteria containing both F and lambda plasmids, both replicate independently, and elimination by growth in acridine orange is also independent. Carriers of lambda Nsus plasmids are not immune, and there is complementation between the plasmids and superinfecting lambda mutants.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli.

We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end). These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.     

Cloning and biological characterization of the immunity region of Escherichia coli phage Mu.

The construction of three hybrid plasmids containing different parts of the left or immunity and end of phage Mu DNA is described. The recombinant plasmids pKN05 and pKN54 carry the HindIII.C and PstI.C fragments of Mu DNA, respectively. Neither of these plasmids expresses the killing function. Moreover, they do not allow plating of superinfecting Mu phages. Plasmid pKN62 harbors the fragment located in between the left PstI and EcoRI cleavage sites on Mu DNA, allows plating of superinfecting Mu phages, but does not express the killing function. These data suggest that the gene coding for the killing function is either positively regulated by a product from the EcoRI.C fragment, or the killing function requires a second product not coded for by pKN62. Mu Vir A- or Mu Vir B- phages are able to grow on bacteria harboring the recombinant plasmid pKN001 which carries the left and EcoRI-C fragment of Mu DNA. This indicates that the superinfecting phages can induce the corresponding gene functions from pKN001. No such induction could be detected in cells harboring the hybrid plasmids pKN05, pKN54 or pKN62.     

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

Isolation and characterization of a rolling-circle-type plasmid from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant-protein expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a theta-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Phenotypic Consequences of Altering the Copy Number of abiA, a Gene Responsible for Aborting Bacteriophage Infections in Lactococcus lactis

The abiA gene (formerly hsp) encodes an abortive phage infection mechanism which inhibits phage DNA replication. To analyze the effects of varying the abiA gene dosage on bacteriophage resistance in Lactococcus lactis, various genetic constructions were made. An IS946-based integration vector, pTRK75, was used to integrate a single copy of abiA into the chromosomes of two lactococcal strains, MG1363 and NCK203. In both strains, a single copy of abiA did not confer any significant phage resistance on the host except for one of the MG1363 integrants, NCK625, which exhibited a slightly higher level of resistance to phages sk1 and p2. Hybridization of the total cellular RNA from NCK625 to an abiA-specific probe indicated that the integration took place downstream of a promoter causing stronger expression of abiA in this integrant. Three abiA-containing plasmids of various copy numbers were introduced into both strains, and the recombinants were evaluated for resistance to phages c2, p2, sk1, and phi31. Plasmid pTRK18 has a copy number of approximately six (cn = 6) and caused a decreased plaque size for all phages evaluated. Integration of pTRK75 into a native plasmid of NCK203 generated pTRK362 (cn = 13), which caused a reduced efficiency of plaquing (EOP = 10) and reduced plaque size. A high-copy-number abiA plasmid (pTRK363), based on the pAMbeta1 origin of replication, was also constructed (cn = 100). Plasmid pTRK363 caused a significant reduction in EOP (10 to 10) and plaque size for all phages tested, although in some cases, this plasmid caused the evolution of AbiA-resistant phage derivatives. Altering the gene dosage or expression level of abiA significantly affects the phage resistance levels.     

Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli

Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology. The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline. We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose-limited batch and chemostat cultures. If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid-containing bacteria grow at the same rate.     

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.     

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.

Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.