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.     

Genetic study of the effect of lambda phage on Mu phage production in Escherichia coli K-12 strains with Mu--lambda--Mu structures

The interaction of temperate bacteriophages Mu and lambda is studied during their simultaneous induction in specially constructed heterolysogenic strains of Escherichia coli bearing trimeric Mu--lambda--Mu structures. These strains were obtained by the MU-mediated integration of phage lambda circular genomes. Heterolysogenic strains of E. coli were used for studying phage lambda eliminating effect on Mu development with a simultaneous induction of prophages in the same cell. The results of the study allow the localization of the region of phage lambda genome incorporating gene (genes) lambda, which produces an eliminating effect on Mu development.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.     

Characterization of a second lysine decarboxylase isolated from Escherichia coli.

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.     

Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr'' prophage.

Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.     

Isolation and characterization of plaque-forming lambdadnaZ+ transducing bacteriophages.

The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with lamdacI857 integrated at a secondary attachment site near purE, transducing phages (lambdadnaS+) that transduced a dnaZts (lambda+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the lambda DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i,e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of lambda DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed lambda DNA between the points 39.3 to 66.5% of lambda length (measured in percent length from the left and of lambda phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard lambda transducing phages are deleted for one prophage end only. In lambdagal and lambdabio strains, the deletions of lambda DNA begin at the union of prophage ends (i.e., position 57.3% of lambda length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A, D. Hershey [ed.], The Bacteriophage Lambda, p. 45-82, 1971). Models for formation of the lambdadnaZ+ phages are discussed.     

Mapping of insertion mutations in gnd of Escherichia coli with deletions defining the ends of the gene.

A genetic map was prepared for gnd, the gene of Escherichia coli which encodes the metabolically regulated 6-phosphogluconate dehydrogenase. Direct selection methods were used for the isolation of mutants with deletions that define the respective ends of gnd. These selections depended on the availability of a defective lysogen in which gnd was present on a lambda h80 dgnd his prophage located at the att phi 80 region of the chromosome. Mutants with deletions entering gnd from the his-distal end were selected as Gnd- TonB- mutants. Mutants with his-proximal gnd deletions were selected as Gnd-, temperature-resistant mutants of a specially prepared stable lysogen. Gnd- mutants were also isolated after mutagenesis with bacteriophage Mu cts61, and genetic tests were used to determine which mutants carry a Mu cts61 prophage in gnd. The deletion mutations were mapped against each other and against the insertion mutations through the use of F' merodiploid strains. The insertion mutations mapped at seven distinct sites in gnd; three mapped under the deletions defining the his-proximal portion of the gene and three mapped with the his-distal deletions.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12.

Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein. A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21. The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC). These mutations, as expected, mapped in the 9.8-min region of the E. coli chromosome by P1 transduction (16% linkage to proC+). Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication. All the mutant strains had growth rates identical to that of wild-type E. coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization. The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.     

Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

Occurrence of the bacteriophage lambda receptor in some enterobacteriaceae.

In Escherichia coli K-12, the receptor for phage lambda is an outer membrane protein which inactivates the phage in vitro. Lambda receptor activity was found in extracts from all wild strains of E. coli tested, although most of them fail to support growth of the phage. In some cases this failure is due to a masking of the receptor in vivo, the bacteria being unable to adsorb the phage or to react with antireceptor antibodies. In other cases, adsorption does occur, and the nature of the block in phage growth was not investigated. Most Mal+ strains of Shigella have lambda receptor, whereas most Mal- strains do not have it. Synthesis of the lambda receptor in Shigella is thus presumably controlled by the positive regulator gene of the maltose regulon as is the case in E. coli K-12. Phage lambda adsorbs on many Mal+ strains of Shigella and even yields plaques on some of them, although at a low frequency. No lambda receptor activity could be found in extracts of several strains of Salmonella and Levinea.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Genetic analysis of the rnc operon of Escherichia coli.

RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes. Clones of the region of the E. coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in lambda with DNA known to map near rnc. A phage clone with the rnc region was randomly mutagenized with a delta Tn10 element, and the insertions were recombined onto the chromosome, generating a series of strains with delta Tn10 insertions in the rnc region. Two insertions that had Rnc- phenotypes were located. One of them lay in the rnc gene, and one was in the rnc leader sequence. Polarity studies showed that rnc is in an operon with two other genes, era and recO. The sequence of the recO gene beyond era indicated it could encode a protein of approximately 26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression. Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E. coli growth but that rnc and recO are dispensable.     

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.