Reproductive fitness of P1, P2, and Mu lysogens of Escherichia coli.

P1, P2, and Mu lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats. Thus, prophage-containing stains of E. coli are reproductively more fit than the corresponding nonlysogens. If mixed populations are grown by serial dilution under conditions in which growth is not limited, both the lysogen and nonlysogen manifest identical growth rates. The increased fitness of the lysogens in glucose-limited chemostats correlates with a higher metabolic activity of the lysogen as compared with the nonlysogen during glucose exhaustion. We propose that P1, P2, Mu, and lambda prophage all confer an evolutionarily significant reproductive growth advantage to E. coli lysogenic strains.     

Stationary phase-like properties of the bacteriophage λ Rex exclusion phenotype

The rex genes of bacteriophage lambda were found to protect lysogenic Escherichia coliK host cells against killing by phage T4 rII, when compared in parallel to isogenic Rex(-) lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex(+) lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. These phenotypes were accentuated in nonlysogenic cells carrying multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous. Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII.     

Wild-type bacteriophage T4 is restricted by the lambda rex genes.

The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens. The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well. Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex.     

Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system

Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.     

Modelling the stability of Stx lysogens

Shiga-toxin-converting bacteriophages (Stx phages) are temperate phages of Escherichia coli, and can cause severe human disease. The spread of shiga toxins by Stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. Lysogens of Stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. We have developed a mathematical model to examine whether known differences in operator regions and binding affinities between Stx phages and lambda phage can account for the lower stability of Stx lysogens. The Stx phage 933W has only two binding sites in its left operator region (compared to three in phage lambda), but this has a minimal effect on 933W lysogen stability. However, the relatively weak binding affinity between repressor molecules and the second binding site in the right operator is found to significantly reduce the stability of its lysogens, and may account for the hair-trigger nature of the switch. Reduced lysogen stability can lead to increased frequency of genetic recombination in bacterial genomes. The development of the mathematical model has considerable utility in understanding the behaviour and evolution of the molecular switch, with implications for phage-related diseases.     

Inhibition of transformation in Streptococcus pneumoniae by lysogeny.

Streptococcus pneumoniae R6X was lysogenized with bacteriophage 304 isolated after mitomycin induction of an ungrouped alpha-hemolytic streptococcus. Lysogenized pneumococci lost their capacity to undergo genetic transformation: transformability was restored after cells were spontaneously cured of their prophage. Both lysogens and nonlysogens produced activator substance (competence factor), and both bound deoxyribonucleic acid in a deoxyribonuclease-resistant form. However, nonlysogens retained deoxyribonucleic acid after washing, whereas lysogens did not. The latter did not liberate phage nor (unlike nonlysogens) degrade transforming deoxyribonucleic acid and contained normal levels of endonuclease.     

Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine.

Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.     

Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype

The sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to Rex exclusion by lambda rexA+ rexB+ lysogens is modulated by the prophage cI repressor allele. We show the following: (i) lambda spi156 delta nin5 forms plaques on a cI+-rexA+-rexB+ lysogen with 10(5)-fold higher efficiency than on cI[Ts]-rexA+-rexB+ derivatives. (ii) The cI[Ts]857 allele augmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involves the participation of a genetic element mapping outside of cI-rexA-rexB.     

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.     

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.     

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.     

Analysis of the phase variation in lambda reduced immunity lysogens

Summary Two distinct phases characterized by different levels of immunity that appear in some E. coli strains lysogenic for reduced immunity mutants of bacteriophage lambda are identified as single and tandem double lysogens respectively on the basis of DNA-DNA hybridization experiments and the requirement of the phage xis function for the transition from a single to a double, and of the host recA function for the transition from a double to a single lysogen (in a xis ^- condition). Rim lysogens with a further increase in immunity, containing some 5 copies of the lambda genome per host genome, have also been observed.It is argued that the different levels of immunity are a direct reflection of the CI gene dosage effect.An unexplained finding is that rim single lysogens yield double lysogens with a frequency of near 1% per generation, whereas cured cells fail to appear even at a frequency 100 times lower.     

Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-phi 1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as phi CP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage phi CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some phi CP6-1 phage up to day 137, yet hardly any phi CP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.     

Isolation and characterization of lambda pleu bacteriophages.

In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage lambda into cistron leuA. The orientation of lambda in the chromosome is ara leuDCB lambda JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., lambda pleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., lambda pleu 13) transduces leuA strains to prototrophy. lambda pleu 13 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 lambda pleu13). A phage carrying the entire leucine operon (lambda K2) was constructed by a cross between lambda pleu9 and lambda pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with lambdaK2 indicated that leuO and leuP are present and functional in lambda K2. leu-specific messenger ribonucleic acid from E. coli hybridizes to the heavy (r) strand of lambdaK2. The leucine operon of lambda G4 pleuABCD (an S7 derivative of lambda K2) exists intact on a 7.3 x 10(6)-dalton fragment (lambdaG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between lambda G4 and lambda show a 5.4 x 10(6)-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 10(6)-dalton piece of lambda DNA starting at 0.46 fractional unit on the map of lambda. Fragment lambda G4EcoRI-B has about 0.6 x 10(6) daltons of lambda DNA from the b2 region at one end and about 1.4 x 10(6) daltons of lambda DNA from the int region at the other end.     

Seasonal Population Dynamics and Interactions of Competing Bacteriophages and Their Host in the Rhizosphere

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-Φ1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as ΦCP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage ΦCP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some ΦCP6-1 phage up to day 137, yet hardly any ΦCP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Excision of bacteriophage lambda from a site in the arabinose B gene.

A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.