Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO.

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.     

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli.

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.     

Transactivation of p'R promoter of phage lambda

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.     

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.     

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module

We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.     

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112

D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

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.     

The role of prophage for genome diversification within a clonal lineage of Lactobacillus johnsonii: characterization of the defective prophage LJ771

Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (“antitoxin”)/pemK (“toxin”) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5′ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.